RADIATION OR OTHER CELL DAMAGE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of US Provisional Application Serial No. 63/390,246, filed July 18, 2022, incorporated by reference in its entirety herein.

GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under grants TR002154, EB027062, CA249799 and CA275733 awarded by the National Institutes of Health and grant 1644869 awarded by National Science Foundation. The government has certain rights in the invention.

BACKGROUND

[0003] Cosmic radiation is a serious risk that will be encountered during missions to the Moon and Mars. There is a compelling need to understand the effects of cosmic radiation, safety thresholds, and mechanisms of tissue damage, in order to develop measures for radiation protection during extended space travel. As animal models fail to recapitulate the exact mutational changes expected for astronauts, engineered human tissues and “organs-on-a-chip” are valuable tools for studying effects of radiation in vitro.

SUMMARY OF THE DISCLOSED SUBJECT MATTER

[0004] A first aspect provides an engineered cardiac tissue comprising induced pluripotent stem cell (iPSC)-derived cardiomyocytes and primary human cardiac fibroblasts encapsulated within fibrin hydrogels stretched between two flexible pillars, matured by electromechanical stimulation at 2Hz, and exposed to radiation or temperatures below -40 °C.

[0005] Embodiments include the following, alone or in any combination.

[0006] The radiation may comprise neutron radiation. The neutron radiation may comprise a proxy for simulated galactic cosmic rays. The radiation may comprise photon radiation.

[0007] The engineered cardiac tissue may be exposed to temperatures below -50 °C.The engineered cardiac tissue may be exposed to temperatures below -79 °C.

[0008] The iPSC-derived cardiomyocytes may comprise a GCaMP reporter.

[0009] The engineered cardiac tissue may be co-cultured with engineered bone marrow tissue. The engineered bone marrow tissue may comprise induced pluripotent stem cells (iPSCs) differentiated into mesenchymal stromal cells (iMSCs) and infused into a decellularized bone matrix scaffold, wherein the iMSCs are thereby differentiated into osteoblasts. The engineered bone marrow tissue may further comprise iMSCs and human umbilical vein endothelial cells (HUVECs) suspended in fibrin hydrogel infused into the bone matrix scaffold. The engineered bone marrow tissue may further comprise cord blood derived CD34 ⁺ hematopoietic cells (HSPCs) infused into the bone scaffold.

[0010] The engineered cardiac tissue may be cultured in a first compartment of a multi-organ tissue bioreactor and the engineered bone marrow tissue is cultured in a second compartment of the multi-organ tissue bioreactor, wherein the first compartment and the second compartment are in fluid communication with a common vascular circulation compartment via an endothelialized barrier between the first compartment and the second compartment.

[0011] A second aspect provides an engineered bone marrow tissue comprising induced pluripotent stem cells (iPSCs) differentiated into mesenchymal stromal cells (iMSCs) and infused into decellularized bone matrix scaffolds, wherein the iMSCs are thereby differentiated into osteoblasts.

[0012] Embodiments of the engineered bone marrow include the following, alone or in any combination. [0013] The engineered bone marrow tissue may further comprise iMSCs and human umbilical vein endothelial cells (HUVECs) suspended in fibrin hydrogel infused into the bone scaffold. The engineered bone marrow tissue may further comprise cord blood derived CD34 ⁺ hematopoietic cells (HSPCs) infused into the bone scaffold.

[0014] The engineered bone marrow tissue may be exposed to radiation. The radiation may comprise neutron radiation. The neutron radiation may comprise a proxy for simulated galactic cosmic rays. The radiation may comprise photon radiation.

[0015] Another aspect provides a method for culturing an engineered cardiac tissue, the method comprising encapsulating induced pluripotent stem cell (iPSC)-derived cardiomyocytes and primary human cardiac fibroblasts within fibrin hydrogels; stretching the fibrin hydrogels between two flexible pillars; maturing the engineered cardiac tissue by electromechanical stimulation at 2Hz; and exposing the engineered cardiac tissue to radiation or temperatures below -40 °C.

[0016] Embodiments of the method include the following, alone or in any combination.

[0017] The radiation may comprise neutron radiation. The neutron radiation may comprise a proxy for simulated galactic cosmic rays. The radiation may comprise photon radiation.

[0018] The method may comprise exposing the engineered cardiac tissue to temperatures below -50 °C. The method may comprise exposing the engineered cardiac tissue to temperatures below -79 °C.

[0019] The iPSC-derived cardiomyocytes may comprise a GCaMP reporter.

[0020] The method may comprise co-culturing the engineered cardiac tissue with engineered bone marrow tissue. The engineered bone marrow tissue may comprise induced pluripotent stem cells (iPSCs) differentiated into mesenchymal stromal cells (iMSCs) and infused into decellularized bone matrix scaffolds, thereby differentiated into osteoblasts. The engineered bone marrow tissue may further comprise iMSCs and human umbilical vein endothelial cells (HUVECs) suspended in fibrin hydrogel infused into the bone scaffold. The engineered bone marrow tissue may further comprise cord blood derived CD34 ⁺ hematopoietic cells (HSPCs) infused into the bone scaffold.

[0021] The engineered cardiac tissue may be cultured in a first compartment of a multi-organ tissue bioreactor and the engineered bone marrow tissue is cultured in a second compartment of the multi-organ tissue bioreactor, wherein the first compartment and the second compartment are in fluid communication with a common vascular circulation compartment via an endothelialized barrier between the first compartment and the second compartment.

[0022] Another aspect provides a method for culturing an engineered bone marrow tissue comprising obtaining induced pluripotent stem cells (iPSCs) differentiated into mesenchymal stromal cells (iMSCs); infusing the iMSCs into a decellularized bone matrix scaffold, thereby differentiating the iMSCs into osteoblasts; and exposing the engineered bone marrow tissue to radiation.

[0023] Embodiments of the method include the following, alone or in any combination.

[0024] The method may further comprise infusing iMSCs and human umbilical vein endothelial cells (HUVECs) suspended in fibrin hydrogel into the bone matrix scaffold. The method may further comprise infusing cord blood derived CD34 ⁺ hematopoietic cells (HSPCs) into the bone matrix scaffold.

[0025] The radiation may comprise neutron radiation. The neutron radiation may comprise a proxy for simulated galactic cosmic rays. The radiation may comprise photon radiation.

[0026] Another aspect provides a bioengineered bone marrow model comprising osteoblasts, mesenchymal stem cells or mesenchymal stromal cells, endothelial cells, and hematopoietic stem and progenitor cells (HSPCs).

[0027] Embodiments include the following, alone or in any combination.

[0028] The iPSC-derived component cells may be encapsulated in fibrin or matrix-derived hydrogels and infused in a decellularized bone scaffold.

[0029] The bioengineered bone marrow model may comprise primary human cell sources comprising primary mesenchymal stem cells, endothelial cells, and cord blood- and bone marrow-derived CD34+ hematopoietic stem/progenitor cells.

[0030] The endothelial cells may comprise human umbilical vein endothelial cells. [0031] The bioengineered bone marrow model may comprise healthy stroma of the endosteum (osteoblasts, MSCs), healthy cells of the perivascular niche (endothelial cells, MSCs), healthy bone marrow or cord blood-derived HSPCs..

[0032] The stroma of the endosteum may comprise osteoblasts, mesenchymal stem cells or combinations thereof.

[0033] The cells of the perivascular niche may comprise endothelial cells, mesenchymal stem cells or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figures 1 A-1E show aspects of an experimental design for engineered human tissue models for assessing the effects of galactic cosmic rays, according to an exemplary embodiment.

[0035] Figures 2A-K show aspects of characterization of engineered human tissues, according to exemplary embodiments.

[0036] Figures 3A-F show evidence that acute radiation causes structural, functional, and molecular changes to engineered cardiac muscle tissues, according to exemplary embodiments.

[0037] Figures 4A-F show evidence that ionizing radiation causes changes to bone marrow microenvironment, according to exemplary embodiments.

[0038] Figures 5A-5O show aspects of singlc-ccll transcriptomics revealing myeloid skewing in irradiated eBM groups, according to exemplary embodiments.

[0039] Figures 6A-G show that differential expression analysis of irradiated neutron eBM tissues reveals increased matrix remodeling phenotype, according to exemplary embodiments.

[0040] Figures 7A-F show aspects of identification of unique phenotypes emerging in eBM at three weeks post-irradiation, according to exemplary embodiments.

[0041] Figures 8A-K show aspects of gene expression over pseudotime and inferred cell-cell, cell-ECM, and cell-secretome interactions confirming myeloid matrix-remodeling phenotype in irradiated eBM cells, according to exemplary embodiments.

[0042] Figures 9A-I show aspects of parallel analysis of photon and mixed neutron sources of radiation for eCM and eBM tissues, according to exemplary embodiments.

[0043] Figures 10A-E show aspects of sample flow cytometr gating for suspension cells in engineered bone marrow model, according to exemplary embodiments.

[0044] Figures 11A-E show exemplary structural and functional cardiac changes associated with radiation exposure, according to exemplary embodiments. [0045] Figures 12A-E show aspects of cardiac transcriptomic changes, according to exemplary embodiments.

[0046] Figures 13A-E show aspects of cardiac molecular changes associated with photon radiation compared to a healthy control, 3 weeks post-radiation, according to exemplary embodiments.

[0047] Figures 14A-C show aspects of characterization of eBM at varied doses of radiation, according to exemplary embodiments.

[0048] Figures 15A-L show classification of marker groups and dendrogram of each cluster in different irradiation conditions, according to exemplary embodiments.

[0049] Figures 16A-T show classification of marker groups with top 20 differentially expressed genes, according to exemplary embodiments.

[0050] Figures 17A-J show differential expression of key hematopoietic groups in response to radiation, according to exemplary embodiments.

[0051] Figures 18A-D show differential expression of key hematopoietic groups in response to radiation, according to exemplary embodiments.

[0052] Figures 19A-C show differential expression of key hematopoietic groups in response to radiation, according to exemplary embodiments.

[0053] Figures 20A-C show inferred incoming and outgoing interactions within 1 Gy group via CcllChat, according to exemplary embodiments.

[0054] Figures 21 A-E show additional differentially expressed changes associated with eCM tissues and their response to 4 Gy Photon versus 1 Gy Neutron, according to exemplary embodiments.

[0055] Figures 22A-M show aspects of using engineered human tissues for therapeutic testing of radioprotective agents, according to exemplary embodiments.

[0056] Figures 23A-D show aspects of recruitment of eBM-derived blood and immune cells in a multiorgan chip system, according to exemplary embodiments.

[0057] Figures 24A-I show aspects of eBM-released CD45+ blood cells in circulation and on endothelial barriers 10 days post-injury, according to exemplary embodiments.

[0058] Figures 25A-L show aspects of cytokine responses to cryoinjury, according to exemplary embodiments.

[0059] Figures 26A-I show aspects of velocity analysis, according to exemplary embodiments.

DETAILED DESCRIPTION [0060] To address the need to understand the effects of cosmic radiation, safety thresholds, and mechanisms of tissue damage, we have developed a bioengineered tissue platform in which we can study radiation damage in individualized or patient-specific settings. Here we describe studies of radiation effects using two engineered tissues: engineered cardiac tissues (eCT, a target of chronic radiation damage) and engineered bone marrow (eBM, a target of acute radiation damage. We report the effects of high-dose neutrons as a proxy for simulated galactic cosmic rays, identification and expression of key genes implicated in tissue responses to ionizing radiation, phenotypic and functional changes in both tissues and proof-of-principle application of radioprotective agents. Our findings identify key changes to inflammatory, oxidative stress, and matrix remodeling genes in both BM and CM, and found that development of an early hypertrophic phenotype in CM and myeloid skewing in BM. Gene expression patterns in BM-derived myeloid cells are significantly altered in irradiated hematopoietic cells. Studying cosmic radiation using individualized models of human organs may give insight into radiation risks in future deep-space missions and whether such health risks may be mitigated using novel radioprotective agents or measures.

[0061] Foran anticipated deep space mission to Mars, NASA has identified “red risks” as areas of concern to human health, including the effects of space radiation on critical organs of the body. Notably, animal models that are routinely used to assess the effects of radiation do not recapitulate the effects seen in humans. Galactic cosmic rays (GCR) primarily comprise protons, followed by alpha particles, and include a small (<1%) fraction of high charge and energy (HZE) particles. Secondary radiation fragments are produced when primary components of GCR interact with target material, such as spacecraft or human tissue, leading to production of secondary protons and neutrons, combining to form a population of high- linear energy transfer (high-LET) sources of radiation. While HZE particles are only a small fraction of GCR, they are a component that is most damaging to human tissues, resulting in clustered damage, including wide tracks of damage when passing through the cells. Disclosed herein are our studies modeling the effects of this most damaging fraction of cosmic radiation using “organs-on-chip” (OoC) human tissue platforms.

[0062] Radiation exposure is associated with a host of acute and chronic symptoms - including prolonged, later-in-life occurrence of cancers and cardiovascular disease (CVD). Most notorious of the short-term effects of radiation exposure is acute radiation syndrome (ARS), which is caused by acute exposures to >1 Gy of radiation and can be broken down into hematopoietic, gastrointestinal, and cerebrovascular aberrations or sub-syndromes, which have significant overlap in symptoms and cover immediate effects in target radio-sensitive organs. In an anticipated ~3-year Mars mission, there could be an approximately 300 to 450 mGy cumulative dose to astronauts, spread over the entire space travel and planet-level habitation. Such exposures to GCRs, particularly in the low-dose, protracted radiation settings, may result in an increase in cataracts, cardiovascular disease, cognitive impairment, reproductive issues, and cancer.

[0063] Previous ground-level work on HZE particles revealed unique double- and single-strand breaks within human cell nuclei, increased genome-level mutations and oxidative damage, leading to further epigenetic changes that may contribute to inability to perform DNA repair, aberrant signaling, and development of cancer. In the cardiovascular system, high doses of radiation from cancer therapies have resulted in increased incidence of CVD and decline in functionality of cardiovascular tissues, including atherosclerosis and myocardial fibrosis.

[0064] Studies on the effects of high-LET radiation have largely been limited due to the complex logistics and high costs associated with conducting experiments in space. NASA’s Brookhaven National Laboratory has developed a terrestrial galactic cosmic ray simulator (GCRsim) providing seven different ion types with several levels of energy and a series of 33 separate beams. Simpler radiation source systems, comprising one or two high-LET radiation types (i.e mixed neutron, Fe-ion, etc ), have also been used to simulate space radiation with easier accessibility and less experimental constraints.

[0065] The detrimental effects of small HZE particle concentrations from ionizing radiation have only recently begun to be explored in human cells and tissues. Secondary neutron ions from cosmic radiation exposure of spacecrafts regolith on the surfaces of the Moon and Mars are an area of intense interest, as damaging high-LET exposures are the greatest risk for long space missions (such as mission to Mars).

[0066] Previous work has demonstrated dose-dependent effects of neutron radiation on differentially expressed genes, as compared to gamma and proton sources. Over short time periods, such as a few days post-neutron radiation exposure, many groups have reported an immediate increase in circulating inflammatory signals, by serum lipidomic/metabolomic analysis or circulating blood cell counts. Many early studies are limited to small animal and monolayer cell culture models. Since human tissues and animal tissues respond differently to radiation damage, especially with respect to immune responses to injury and repair, experimental data in mice and other animal models have had limited translational use.

[0067] Over the past decade and in response to the limitations in drug development using animal models, engineered human tissue models, including organoids and organs-on-a-chip (OOC), have emerged as new tools for investigating disease progression and therapeutic modalities such as the safety and efficacy of drugs. Engineered tissues generated from primary human cells and induced pluripotent stem cells (iPSC) have been used to recapitulate the microenvironmental diversity (i.e. supporting cells, extracellular matrix (ECM) components and architecture) of many human tissues on the micro-scale, mimicking functional features of native human organs. To date, many models have emerged for studies of human (pathophysiology, in the heart, liver, bone marrow, skin, gut, kidney, and brain tissues individually or in combinations, among others. A few groups have recently reported use of engineered models for studying space health for studies of low-LET radiation sources and microgravity, although only during short trips to the International Space Station (ISS), studies at NASA’s Brookhaven National Labs, or with clinical sources of ionizing radiation. None of these studies have begun to characterize the potential changes to the human body amidst exposure to GCRs in deep space to date. As deep space missions are now in planning, there is a pressing need to assess and counter the effects of exposure of human body to deep space radiation. [0068] Here we describe the first use of engineered human tissue models for simulating the effects of GCRs and secondary radiation exposures (mixed neutrons) expected in deep space during a Mars mission. Engineered human cardiac tissue (eCT, selected as a site of long-term radiation damage) and human bone marrow (eBM, selected as a site of acute radiation damage) were formed from cells and tissue-specific extracellular matrix. Both tissues were matured to acquire functional characteristics of the respective native organs and exposed to the types and dosages of radiation relevant to cosmic radiation expected during long space missions. We assessed the effects of radiation over a period of 3 weeks post-exposure, using comprehensive molecular, structural and functional assays.

[0069] Figures 1A-E show aspects of an experimental design for engineered human tissue models for assessing the effects of galactic cosmic rays. Fig. 1A shows a schematic representation of an engineered human bone marrow tissue model and timeline. Fig. IB shows a schematic representation of an engineered human cardiac muscle tissue model and timeline. Fig. 1C shows a schematic representation of the project overview. Fig. ID shows images of neutron beam accelerators at Columbia University’s Radiological Research Accelerator Facility (RARAF) facility. Fig. IE shows another schematic representation of the project overview in which engineered tissues are exposed to acute doses of photons or neutron irradiation. [0070] Using engineered models of the human bone marrow (eBM), selected as a site of acute radiation damage, and cardiac muscle (eCT), selected as a site of prolonged and chronic radiation damage, we designed and matured each tissue to mimic a subset of functional characteristics of the respective native organ (Figs. 1A-B). eBM tissues were derived by infusing a combination of osteoblasts, mesenchymal stem/stromal cells (MSCs), endothelial cells, and cord blood-derived hematopoietic stem/progenitor cells (HSPCs) into decellularized bone scaffolds. eCT tissues were derived by encapsulation of human iPSC- derived cardiomyocytes and primary fibroblasts into fibrin hydrogel stretched between two flexible pillars and matured over a period of 4 weeks by electromechanical stimulation.

[0071] After tissue formation and maturation (4 weeks for eCTs and 5 weeks for eBMs), the tissues were exposed to an acute dose of either 4 Gy of photons or 1 Gy of mixed neutrons at Columbia University’s Radiological Research Accelerator Facility (RARAF). We irradiated eCTs and eBMs with (z) mixed neutrons (1 Gy acute dose, approximately ~15 to 17% concomitant photons) or (zz) photons (4 Gy acute dose, with and without radiation protective agents. In addition, healthy tissues with 0 Gy exposure were used as a control (Fig. 1C).

[0072] After 3 weeks of tissue culture post-radiation exposure, we exposed tissue to radiation and studied the structural, functional, and molecular changes associated with radiation damage. Using single-cell sequencing, we were able to identify the emergence of unique myeloid populations and myeloid lineage skewing in irradiated eBMs. Changes to eCT and eBM tissues were compared to published studies of accidental radiation exposures in cancer and nuclear warfare, samples from astronauts returning to Earth, and ex vivo and animal studies of high-LET exposure. We believe that this work lays a foundation for using engineered human tissue models to study the acute and chronic effects of GCRs, giving insight into anticipated risks associated with deep space travel, and testing radioprotective measures.

RESULTS

[0073] Engineered eBM and eCT models mimic human physiological functions

[0074] Our goal was to design easy-to-use, reproducible models of CM and BM derived from a combination of human primary and 1PS cells. To establish the eBM tissue model, we first engineered bone by differentiating iPSC-derived mesenchymal stromal cells (iMSCs) infused into decellularized bone matrix into osteoblasts, over a period of 4 weeks we first engineered bone by infusing iPSC-derived mesenchymal stromal cells (iMSCs) into decellularized bone matrix scaffolds, to induce their differentiation into osteoblasts, over a period of 4 weeks (Fig. 1A). Then we infused iMSCs and human umbilical vein endothelial cells (HUVECs) suspended in fibrin hydrogel into the forming bone, followed by sequential seeding of cord blood derived CD34“ hematopoietic cells (HSPCs). We confirmed the presence of key stromal/hematopoietic markers, including bone sialoprotein (BSP), CXCL12, and CD45 (by immunostaining). We further confirmed key morphological features by pentachrome and Wright- Giemsa staining, as well as micro-computed tomography (pCT) (Fig. 2A).

[0075] Over a period of two weeks after introducing HSPCs into the eBM tissue, we validated the maintenance of CD34+ HSPCs (Fig. 2B), as well as cell differentiation into myeloid subpopulations, including monocytes (CDl lb+, CD14+), dendritic cells (CDl lc+), and granulocytes (CD16+) (Fig. 2C). We observed inverse relationships between the maintenance and expansion of CD34+ cells and the increase in myeloid progenitors overtime (Fig. 2C). Unlike other studies, we cultured the eBMs without adding high concentrations of hematopoietic cytokines (such as stem cell factor, SCF, thrombopoietin, TPO: FMS-like tyrosine kinase 3, FLT3), and instead relied on the endogenous signals produced by the stromal cells. The model supported the natural differentiation trajectory of primary CB-de rived HSPCs in culture, as shown by flow cytometry analysis revealing the production of CDl lb+ cells by day 12 (Figs. 10A-E). We confirmed the presence of multipotent in eBMs after 2 weeks of culture at low cytokine concentrations (5 ng/mL SCF/TPO/FLT-3L), after introduction of CD34+ HSPCs, with approximately 10% of colony forming CD45+ cells at Day 6, and 3% at Day 12 (Fig. 2D-2E).

[0076] We previously published our work in modeling human eCT tissues, showing that iPSC-derived cardiomyocytes and primary human cardiac fibroblasts within fibrin hydrogels and between two flexible pillars could be incrementally matured by electromechanical stimulation over four weeks in culture (Fig. IB). To engineer human cardiac tissues (eCT), we followed our previously published methodology where iPSC-derived cardiomyocytes and primary human cardiac fibroblasts are encapsulated in fibrin hydrogel stretched between two flexible pillars (Figs. 1A, IE, 2G and 2H). The resulting cell-hydrogel constructs were matured by electromechanical conditioning at 2 Hz, over a total of four weeks of culture, towards human tissue phenotype. In some embodiments, tissues were made using iPSC-derived cardiomyocytes with a GCaMP reporter, to allow for longitudinal online analysis of tissues by bright field and calcium fluorescent imaging.

[0077] Figures 2A-J show aspects of characterization of engineered human tissues. Fig. 2A shows histological staining of key hematopoietic support and downstream blood/immune markers. Fig. 2B shows characterization of CB-derived CD34+ cells and CD34+CD38- HSPCs, and further delineations of progenitors (i.e. MPPs, MLPs, CMPs/GMPs), on eBM tissues over 1-2 weeks. Fig. 2C shows that HSPCs on human eBM begin to differentiate into myeloid cells (i.e. monocytes, dendritic cells, granulocytes) over 1-2 weeks. Figs. 2D and 2E show characterization of multipotent potential of HSPCs over 1-2 weeks using colony forming unit assays. Fig. 2F shows reactor platforms to make engineered cardiac muscle tissues. Fig. 2G shows an example bright field image of engineered heart tissues on flexible pillars. Fig. 2H shows example images of aligned cardiomyocytes stained for a-actinin (magenta), cardiac troponin (green), and DAPI (blue); scale = 20 p). Fig. 21 shows increasing force-frequency relationship of cardiac muscle tissues. Fig. 2J shows an example calcium trace of eCM tissues. Fig. 2K shows responses of eCTs to isoproterenol, a beta-adrenergic agonist. Data shown as mean +/- SD. *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. [0078] We confirmed the hallmarks of tissue maturation, including cell alignment, striations (Fig. 2H), positive force/frequency relationship (Fig. 21), and contractile responses to adrenergic stimulation (Fig. 2J). [0079] For eBM tissues, we confirmed the presence of key stromal/hematopoietic markers, including bone sialoprotein (BSP), SDFla/CXCL12, and CD45, along with key morphological markers (pentachrome, Wright-Giemsa, micro-computed tomography (uCT)) (Fig. 2A). Over a period of two weeks after introduction of HSPCs into the eBM tissue, we validated the maintenance of CD34 ⁺ HSPCs in the eBM tissues (Fig. 2B) and differentiation into myeloid subpopulations, including monocytes, dendritic cells, and granulocytes (Fig. 2C). Consistent with other studies in the field, we showed an inverse relationship with maintenance and expansion of CD34 ⁺ cells and increase in differentiating myeloid progenitors overtime (Figs. 2B-2C). Unlike other studies, we designed the model with endogenous signals produced by the stromal cells that drive natural hematopoietic differentiation, without addition of high concentrations of hematopoietic maintenance cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 (FLT3). The model supported a natural differentiation trajectory of primary CB-derived HSPCs in culture as summarized in Figs. 10A-E.

[0080] Figures 10A-E show aspects of sample flow cytometry gating for suspension cells in our engineered bone marrow model. Figs. 10A-B show myeloid flow cytometry panels with shifting populations over time (Fig. 10A) and dimensionality reduction (Fig. 10B) for CD14 and other markers (CD14, CDl lb, CDl lc, CD16, CD15). Figs. 10C-D show hematopoietic flow cytometry panel with shifting populations over time (Fig. 10C) and dimensionality reduction (Fig. 10D) for CD34 and other markers (CD34, CD38, CD45RA, CD90). Fig. 10E shows subpopulations of hematopoietic progenitors attached to bone tissue and in suspension.

[0081] We confirmed the presence of multipotent progenitors in eBMs after 2 weeks of in vitro culture at low cytokine concentrations (5 ng/mL SCF/TPO/FLT-3L) after introduction of CD34 ⁺ HSPCs, with approximately -10% of colony forming CD45 ⁺ cells at Day 6, and -3% at Day 12 (Figs. 2D-2E).

[0082] For eCT tissues, we confirmed the hallmarks of tissue maturation, including a positive forcefrequency relationship (Figs. 2F-2I), and stable calcium transients in response to stimulation (Fig. 2 J). After tissue formation and maturation (roughly 4 weeks for eCT and 5 weeks for eBM models), tissues were acutely exposed to either 4 Gy of photons or 1 Gy of mixed neutrons and studied for a period of three weeks post-radiation. [0083] Effects of acute radiation injury on cardiac tissues (eCT)

[0084] Three weeks after acute radiation exposure with 1 Gy of neutrons (Fig. IB), eCT tissues were evaluated for the presence of cell-type specific markers including cTnT and a-actinin for cardiomyocytes and vimentin for cardiac fibroblasts.

[0085] Figures 3A-G show evidence that acute radiation causes structural, functional, and molecular changes to engineered cardiac muscle tissues Fig. 3A shows immunofluore scent, whole-mount staining of eCM tissues 3-weeks post-irradiation with cardiac Troponin T (cTnT), alpha-actinin, vimentin, and DAPI (scale = 10 um). Fig. 3B shows eCM tissue contractility metrics, including full-width half-max (FWHM), contraction length at 50 and 90 of peak, and relaxation length at 50 and 90 of peak, which was measured with bright field imaging and computation analysis. Fig. 3C shows eCM tissue excitability and conduction metrics, including maximum beating frequency, maximum capture rate, and excitation threshold, which was measured with calcium imaging via a GCaMP reporter line and was normalized to baseline values per tissue. Fig. 3D shows eCM tissue force generation metrics, including passive and active stress, as well as contraction and relaxation velocities, measured with bright field imaging. Figure 3E shows volcano plots showing differentially expressed genes between the irradiated and control eCTs, by RNA sequencing and a comparison of significant differentially expressed genes. Fig. 3F shows biological pathways associated with significant genes in the control and neutron irradiated groups. Data shown as mean +/- SD. *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0086] Neutron-irradiated eCTs demonstrated increased presence of collagen 1 (by immunofluorescence), compared to healthy and 4 Gy photon group (Fig. 3A). Functional analysis was performed for all eCT tissues before radiation and over 3 weeks post-irradiation, using a combination of bright field and fluorescent calcium imaging (based on the endogenous GCaMP reporter) to reveal tissue contractility, excitability, and force generation by measuring muscle movement and pillar displacement. [0087] Immunofluorescent staining indicated a similar presence of both cell types in the microtissues. Striations were present in cardiomyocytes of irradiated groups, but the alignment of irradiated tissues was dysregulated as compared to the non-irradiated controls (Fig. 3A).

[0088] Contractility analysis showed differences in both contraction and relaxation (Fig. 3B). Irradiated tissues had a greater full width half maximum (FWHM) than control tissues (p<0.001), and required longer times for relaxation (p<0.0001, Fig. 3B). There was a significant change in contraction and relaxation lengths between tissues irradiated by photon and neutron sources (p<0.05) (Fig. 3B). Irradiation also caused significant changes in excitability and conduction metrics, including the decreased maximum capture rate and increased excitation thresholds (Fig. 3C). Notably, both the active stress and contraction velocity of neutron-irradiated tissues increased, indicating greater force generation (Fig. 3D).

[0089] Figures 11A-E show exemplary structural and functional cardiac changes associated with radiation exposure. Fig. 11A shows that 1-hour post-acute radiation exposure reveals trends in increased reactive oxygen species (ROS) via R0M01 and mitochondrial biogenesis via SDHA expression. Fig. 1 IB shows histological trichrome staining of cardiac muscle in response to radiation 3-weeks post-exposure. Fig. 11C shows an example pipeline for extracting cardiac functionality metrics of excitation threshold and maximum capture rate. Fig. 1 ID shows example eCM traces 3-weeks post exposure. Fig. 1 IE shows nonsignificant differences between eCM tissue widths at 3 weeks post-irradiation.

[0090] Histological analysis did not show evidence of fibrosis 3 weeks post-irradiation, which is seen following late-stage radiation exposures (Fig. 11B). Functional analysis was performed for all eCT tissues at Day 0 before irradiation, as well as at 3 weeks post-irradiation, using a combination of bright field and fluorescent calcium imaging (endogenous GCaMP reporter) and custom-made and published muscle functionality pipelines (Fig. 11C-E).

[0091] Irradiated tissues demonstrated significant changes to excitability and conduction metrics, including decreased maximum beating frequency, decreased maximum capture rate, and increased excitation thresholds, indicating an increased stimulation frequency necessary to contract the cCT tissues (Fig. 3B). Notably, contractility analysis displayed differences in the timings of contraction and relaxation (Fig. 3C). Irradiated tissues, and most significantly neutron-irradiated tissues, had a greater full width half maximum (FWHM) as compared to control tissues (p<0.001) (Fig. 3C). This same effect was insignificant when only considering the contraction length but was largely significant when looking at the relaxation time of irradiated tissues as compared to the controls (p<0.0001) (Fig. 3C). There was a significant change in contraction length between tissues irradiated by photon and mixed neutron sources (p<0.05), suggesting different mechanisms for the two types of radiation (Fig. 3C). Notably, the force generation of neutron- irradiated tissues, and not both irradiated eCT tissue groups, was significantly higher than control groups when considering contraction velocity and active stress (Fig. 3D). Although there were differences in passive stress and relaxation velocity between groups, these effects were not significant.

[0092] Mixed neutron-exposed eCT tissues revealed differentially expressed genes (p<0.05, log2foldchange >1) compared to controls, including changes to genes implicated in oxidative stress (HM0X1), metabolism (CYP1B1), matrix remodeling (DLK1, NRK, DLL4, COL6A6), cardiac function (TNNT, NRAP, NOV, VASH1) and radiation exposures (CDKN1A). Most notably, HM0X1, a gene implicated in oxidative stress response and mitigation of cytotoxic injury, was significantly increased in neutron-irradiated groups 3 -weeks post-acute exposure (Fig. 3E).

[0093] Gene ontology (GO) pathway analysis revealed upregulation of cardiac-specific pathways of sarcomere organization and striated muscle development in neutron irradiated eCT tissues, as well as increases in apoptosis and phosphorus metabolic pathways (Fig. 3F). Pathway network analysis revealed significant increases in connections between cardiomyocyte -specific pathways and ECM remodeling, indicating reorganization of tissue structure in irradiated groups. Downregulated pathways included blood vessel development and regulation of cell motility (Fig. 3F), indicating a preferential increase in cardiomyocyte-specific pathways as compared to non-myocyte populations in eCT tissues, which may contain up to -15-25% non-myocytes during tissue formation (i.e. fibroblasts, endothelial cells, pericytes, etc).

[0094] Figures 12A-F show aspects of cardiac transcriptomic changes for cardiac-specific functional (Fig. 12A), fibrosis (Fig. 12B), inflammatory (Fig. 12C), DNA damage/repair (Fig. 12D), mitochondrial genes (Fig. 12E), as well as network analysis of differentially expressed genes between neutron and 0 Gy control doses (Fig 12F).

[0095] In analysis of key genes implicated in cardiac muscle function, fibrosis, and inflammation, there was a differential response between photon and neutron sources, notably increased expression of MYH6/MYH7 and IL6/IL1B (Fig. 12A-12C). Photon and neutron sources appeared to have differential effects on DNA damage genes, with increases in CDC25A, RAD 17, BRCA1, and BRCA2 in photon tissues and increases in XRCC3, RAD51, and H2AX in neutron tissues (Fig. 12D, Figs. 5A-O).

[0096] Pathway analysis and cytoscape clustering revealed GO terms associated with muscle development, smooth muscle cell proliferation, matrix organization, and positive regulation of apoptotic processes (Fig. 12F). Only a few of these pathways were found in photon-irradiated tissues (Fig. 13B).

[0097] Figures 13A-E show aspects of cardiac molecular changes associated with photon radiation compared to a healthy control, 3 weeks post-radiation. Fig. 13A shows a principal component analysis plot of samples. Fig. 13B shows bulk gene expression changes in irradiated eCTs three weeks after irradiation illustrating significant differentially expressed genes by fold change for neutron and photon radiation compared to control sCTs. Fig. 13C shows a volcano plot of healthy control vs. photon irradiation. Fig. 13D shows a GO pathway analysis of biological processes. Fig. 13E shows network analysis using Revigo to show connectivity between differentially expressed cardiac genes.

[0098] Effects of acute radiation on bone marrow tissues (eBM) [0099] In parallel to eCTs, we exposed matured eBM tissues to the same acute doses of radiation (either 4 Gy of photons or 1 Gy of neutrons) and studied the effects on their hematopoietic progeny and microenvironment over 3 weeks post-irradiation. Immediately after exposure, we analyzed double -stranded DNA breaks in CD34+ HSPCs, using an ImageStream analysis pipeline to visualize yH2AX in the nuclei of irradiated HSPCs. These data showed early signs of radiation damage, evidenced by the changes in the number and intensity of dsDNA breaks, as well as a dose -dependent effect with increasing dose (Fig. 4A- C, Figs. 14A-C).

[00100J Neutron-irradiated tissues had a larger percentage of cells with 1-3 yH2AX dsDNA foci, compared to photon-irradiated tissues that had a greater spread in the numbers of foci in blood cells (Fig. 4B). Further, there were significant differences in yH2AX foci between all groups (Fig. 4C). Right after radiation exposure, eBM tissues exhibited increased secretion of BM stress factors, including inflammatory cytokines M-CSF and IL-6, stromal-support growth factors CXCL12 and osteopontin (OPN) (Fig. 4D). After 3 weeks of culture, histological analysis confirmed morphological changes in BSP+ osteoblasts, and the decrease in their numbers, without evident changes in tissue-level CXCL12 expression (Fig. 4E). Using flow cytometry, we observed a significant reduction in total blood cell numbers, and an increase in CD45+ median fluorescent intensity (MFI), in irradiated groups (Fig. 4F). The increased CD45+ MFI indicates further skewing towards more differentiated progeny, that was confirmed morphologically by Wright- Giemsa staining of blood cells (Fig. 4E).

[00101] Similarly, eBM tissues were affected by the acute photon and neutron radiation. Three weeks after radiation exposure, eBM tissues had a decreased CD45 ⁺ proliferative capacity, as evidenced by histological staining (pentachrome, BSP) and flow cytometry (CD45 ⁺ expansion, median fluorescent intensity (MFI) of CD45 ⁺ )

[00102] Using the approximation that mixed neutron doses are ~4 times as potent as photon doses, we exposed matured eBM tissues to acute doses of either 4 Gy of photons or 1 Gy of neutrons and studied their hematopoietic progeny and microenvironment 3 weeks post-irradiation. Figures 4A-F show evidence that ionizing radiation causes changes to the bone marrow microenvironment. Fig. 4A shows qualitative example images of DNA-damage marker gH2AX in hematopoietic cells isolated one hour post-radiation. Figs. 4B-C show quantitative analysis in number and average area of dsDNA breaks per cell. Fig. 4D shows that acute doses of radiation cause early signs of inflammation (M-CSF, IL-6) and stromal damage (CXCL12, OPN) in BM tissue culture supernatant. Fig. 4E shows histological staining of BM tissue samples 3-weeks post-radiation treatment. Fig. 4F shows analysis of CD45+ cell proliferation and median fluorescent intensity in samples 3-weeks post-radiation using flow cytometry. *p<0.05, ** p<0.01, *** p<0.001, **** pO.OOOl.

[00103] Immediately after exposure, we analyzed double -stranded DNA breaks in CD34 ⁺ HSPCs, using an ImageStream analysis pipeline to visualize gH2AX in the nuclei of irradiated HSPCs. This data showed early signs of radiation damage, evidenced by the changes in the number and intensity of dsDNA breaks (Fig. 4A-C).

[00104] Neutron-irradiated samples had more cells with 1 -3 gH2AX dsDNA foci, as compared to photon- irradiated samples with a greater spread of foci number in blood cells (Fig. 4B). Further, there was a significant difference in gH2AX foci area between all groups (Fig. 4C). Right after radiation exposure, eBM samples exhibited increased secretion of BM stress factors, including inflammatory cytokines M-CSF and IL-6, as well as stromal-support growth factors CXCL12 and osteopontin (OPN). After 3 weeks in culture, histological analysis confirmed morphological changes in BSP ⁺ osteoblasts, and the decrease in their numbers, without evident changes in CXCL12 expression (Fig. 4E). Using flow cytometry, the total yield of CD45 ⁺ hematopoietic cells was observed, showing a significant reduction in total blood cell number, but an increase in CD45 ⁺ median fluorescent intensity (MFI), in irradiated groups (Fig. 4F). The increased CD45 ⁺ MFI indicates further skewing towards more differentiated progeny, which is confirmed morphologically from Wright-Giemsa staining of blood cells in suspension (Fig. 4E).

[00105] Effects of acute radiation injury on blood cells released from bone marrow tissues (eBM)

[00106] To understand the downstream and long-term changes to radiation exposure, we performed single cell sequencing on 44,078 total CD45 ⁺ cells isolated from the suspension 3 weeks after acute radiation exposure (12,513 in non-radiated controls; 17, 15 in photon irradiated group; 14,050 in neutron irradiated group).

[00107] Figures 5A-0 show aspects of single-cell transcriptomics revealing myeloid skewing in irradiated eBM groups. Fig. 5A shows Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction to identify proposed blood and immune cell progeny after three weeks of culture. Fig. 5B shows a breakdown of proposed UMAP clusters for each experimental condition. Figs 5C-5G show the top three genes per cluster among all conditions. Figs. 5H-I show key example immune markers used for cell type identification. Fig. 5J shows a distribution of major immune cell classifications, showing increased myeloid population, mainly in macrophages, in irradiated groups. Figs. 5K-5O show percentage distributions for each experimental condition within the 18 clusters. [00108] UMAP unsupervised clustering helped identify several subclusters present within our isolated samples (Fig. 5A), as well as the differential responses observed with all conditions, including new clusters emerging during radiation treatment (Fig. 5B).

[00109] Figures 6A-G show that differential expression analysis of irradiated neutron eBM tissues reveals increased matrix remodeling phenotypes. Figs. 6A-E depict violin plots showing increased expression of genes relating to myeloid differentiation (Fig. 6A), matrix degradation (Fig. 6B), cancer (Fig. 6C), radiation (Fig. 6E) and apoptosis (Fig. 6E). Fig. 6F shows top significant, differentially expressed genes in 1 Gy cells compared to the 0 Gy healthy control. Fig. 6G shows pathway analysis of significantly upregulated/downregulated genes in the neutron-irradiated samples in gene ontology biological processes and molecular functions.

[00110] We compared the top differentially expressed genes between individual subclusters and the rest of the cell populations (Fig. 6C), using these top genes in combination with key cell-type specific markers (Figs. 5C through 50) to identify and name different populations seen in Fig. 5A. For simplicity, we looked first at key markers for each cell type: hematopoietic progenitors (CD34, CD43, PR0M1), megakaryocyte progenitors (PPBP, VWF), granulocytes (MPO, ELANE), eosinophils/basophils (CPA3), dendritic cells (CD1C, CD14), lymphoid cells (IL7R), monocytes (CD14, VCAN, ITGAM), and macrophages (CD68, CD 163) (Fig. 5D). Unique cell populations that emerged from this analysis were labeled as subpopulations within a certain group (i.e. Macrophage subpopulation 1 and 2). Using the top genes for each grouping, we identified previously unidentified clusters, including a myeloid-committed cluster (“abnormal myeloid cells”) with high expression of matrix metalloprotease and cell cycle proliferative markers (Figs. 5K-5O Figs. 7A-D) Some populations, like Granulocyte Population 1, may be further specified to neutrophils, as they had high expression of ELANE.

[00111] Comparing irradiated groups to healthy controls, we observed an increase in myeloid cell subtypes (Fig. 6E-F), with a reduction of IL7R ⁺ lymphoid-committed cells and CD1C ⁺ dendritic cells. Of progenitor and lymphoid subtypes, most cells in these groups were from the untreated (non-irradiated), 0 Gy control group, including progenitors (0 Gy control: 80%, photon: neutron), granulocyte -monocyte progenitors, plasmacytoid dendritic cells (v), and lymphoid-committed cells (Figs. 5K-5O).

[00112] Of the myeloid subtypes, there was a large shift in CD14 ⁺ subclusters from monocytes to downstream lineages, differentiated macrophages in irradiated groups, with Monocyte Subpopulation 3 (1.9% in Control, 56.1% in Photons, 42% in Neutrons), Macrophage Subpopulation 1 (2.0% in Control, 53.8% in Photons, 44.2% in Neutrons), Macrophage Subpopulation 2 (8.4% in Control, 58.9% in Photons, 32.8% in Neutrons), and the Abnormal Myeloid Cell (4.0% in Control, 38.9% in Photons, 57.1% in Neutrons) clusters almost exclusively present in the irradiated groups (Figs. 5K-5O and 7A-D).

[00113] In analysis of gene expression of different radiation groups, we found significant increases in key myeloid differentiation markers following 1 Gy neutron exposure compared to the 0 Gy control, including CD14 (p <1O’ ³⁰⁶ , L2FC = 1.48), CD68 (p <10’ ²¹⁴ , L2FC = 0.78), ITGAM (p <1O’ ³⁰⁶ , L2FC = 1.14), VCAN (p <10’ ²⁴ , L2FC = 0.40), and FN1 (p <1O’ ³⁰⁶ , L2FC = 3.44) (Figs. 6A and 7A-D).

[00114] There also was an increase in expression of ECM-associated MMPs in the neutron group, including MMP9 (p <1O’ ³⁰⁶ , L2FC = 3.51) and MMP7 (p <10’ ⁸³ , L2FC = 5.20) (Fig. 6B), increases in cancer-related genes, including DUSP6, traditionally associated with myeloid abnormalities (p <1 O ^-206 , L2FC = 1.67), CD47 (p <0.05, L2FC = 0.06), evasion of tumor cell uptake by immune cells, and TET2 (p<10 ^-40 , L2FC = 0.24), associated with numerous human cancers (Fig. 6C).

[00115] As expected, there was an increase in several radiation-associated genes in the neutron-irradiated group and/or apoptosis, including CDKN2A (p<10 ⁵ , L2FC = 2.80), TP53 (p<10 ⁴⁸ , L2FC = 1.10), and BAX (p<10 ^-4 , L2FC = 0.11) (Fig. 6D). In comparing the neutron-irradiated groups with the control, the highest differentially expressed genes were among those associated with ECM remodeling, including MMP9 and FN1, lipid metabolism, including FBP1, APOE, and G0S2, and macrophage activation/inflammatory- rcsolvc associated genes C1QA and APOCI (Fig. 6E). GO molecular functions revealed an increase in MMP activity and growth factor, proteoglycan and chemokine receptor binding, all associated with ECM degradation by activated myeloid cells (Fig. 5D). This matches closely to the top upregulated GO biological processes, which were associated with ECM remodeling and leukocyte chemotaxis, and the top downregulated pathways, which were associated with immune response and pro-inflammatory signaling (Fig. 6F). GO molecular functions similarly revealed an increase in MMP activity and growth factor, proteoglycan, and chemokine receptor binding, all associated with ECM degradation by activated myeloid cells (Fig. 6F). Notably, all myeloid markers were still increased when compared in single subpopulations (i.e. CD14 ⁺ cells or CD68“ cells) between irradiation groups (Figs. 17A-J, 18A-D, 19A-C).

[00116] Figures 17A-J show differential expression of isolated cells from engineered bone marrow (eBM) single cell RNA sequencing in response to radiation of key hematopoietic groups CD 14+ cells and CD68+ cells. Fig. 17A shows UMAP visualization of CD14+ myeloid cells. Fig. 17B shows differential gene expression in neutron irradiated (1 Gy) tissues relatively to controls. Fig. 17C shows example violin plots of key myeloid genes. Fig. 17D shows GO pathway analysis of top biological processes. Figs. 17E-F show UMAP visualization of CD68+ myeloid cells. Fig. 17G shows differential gene expression in neutron irradiated (1 Gy) tissues relatively to controls. Figs. 17H-I shows example violin plots of key myeloid genes. Fig. 17J shows GO pathway analysis of top biological processes.

[00117] Similarly, Figures 18A-D and 19A-C show differential expression of key hematopoietic groups in response to radiation. Fig. 18A shows UMAP visualization of ELANE+ neutrophils. Fig. 18B shows differential gene expression in neutron irradiated (1 Gy) tissues relatively to controls. Fig. 18D shows example violin plots of key myeloid genes. Fig. 18C shows GO pathway analysis of top biological processes. Fig. 19A shows differential gene expression in neutron irradiated (1 Gy) tissues relatively to controls. Fig. 19C shows example violin plots of key myeloid genes. Fig. 19B shows GO pathway analysis of top biological processes.

[00118] Figures 14A-C show aspects of characterization of eBM at varied doses of radiation. Fig. 14A shows a dose-dependent relationship with eBM histological staining for pentachrome (top) and BSP (bottom). Fig. 14B shows decreasing CD45+ hematopoietic cell production, and Fig. 14C shows increasing MFI of CD45+ cells with increasing radiation dose over two weeks. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05).

[001 19] Figures 15A-L show classification of marker groups and dendrograms of each cluster in different irradiation conditions: 0 Gy Control (Figs. 15A-D); 4 Gy Photon (Figs. 15E-H); 1 Gy Neutron (Figs. 15I-L).

[00120] Figures 16A-I show classification of marker groups with top 20 differentially expressed genes. [00121] Figures 7A-F show aspects of identification of unique phenotypes emerging in eBM at three weeks post-irradiation. Figs. 7A-D show top significant differentially expressed genes and pathways implicated in Macrophage Subpopulation 1 (Fig. 7A), Macrophage Subpopulation 2 (Fig. 7B), Monocyte Subpopulation 3 (Fig. 7C), and Abnormal Myeloid Cells (Fig. 7D). Fig. 7E illustrates MKI67 and MMP9 expression on cell clusters. Fig. 7F depicts pseudotime analysis-inferred cell cycle states, showing unique proliferating capacity of abnormal myeloid cells, compared to the 0 Gy control populations.

[00122] Investigating the emerging populations in neutron-irradiated eBM tissue groups, we saw a shift in myeloid differentiation towards several macrophage phenotypes implicated in matrix degradation and remodeling (Fig. 7A-D). Both the Macrophage Subpopulation 1 (MASI) and Macrophage Subpopulation 2 (MAS2) exhibited characteristics associated with an M2-macrophage polarized phenotype, including expression of key pan- and M2-specific macrophage markers (CD68, CD 163, MS4A4A, PLXDC2, NEAT1), as well as genes implicated in ECM remodeling and microenvironmental metabolism (MMP9, RAL15, RASAL2, CSTB) (Fig. 7A). [00123] Notably, pathway analysis of MAS 1 revealed many signatures of leukocyte migration and ECM organization, as well as lipid catabolic processes. In MAS2, GO processes included pathways related to neural development, morphogenesis, and ECM remodeling, potentially hinting at a pathologic role of these cells in addition to their autophagic phenotype in vitro (Fig. 7B). Monocyte subpopulation 3 (MOS3) expressed many canonical markers of myeloid- and monocyte-specific cell fate, including FCN1, VCAN, and CD 14, but these cells also had high levels of genes associated with myeloid-derived suppressor cell (MDSC) monocytic populations (M-MDSCs), including S100A8, S100A9, and S100A10.

[00124] Pathway analysis for the MOS3 population indicated typical functional processes in immune responses, including myeloid leukocyte activation and neutrophil degranulation, potentially indicating a role in these cells in regulating other immune progeny (Fig. 7C). Most interestingly, there was a population (labeled “abnormal myeloid cells”) that appeared in irradiated groups, exhibiting canonical myeloid markers of CD 14 and CD68, but had significantly higher expression of genes (MKI67, PCLAF, DTYMK) associated with cell proliferation, cell cycle regulation, and DNA repair (Fig. 7D). This unique population also expressed the highest levels of MKI67 in gene expression (Fig. 7E) and cell cycle markers are identified via pseudotime analysis of all cells (Fig. 7F). This group, in addition to MASI and MAS2, also exhibited significantly high levels of MMP9, as compared to all other cells (Fig. 7F).

[00125] Myeloid bias and the emergence of myeloid-derived suppressor populations in neutron-treated eBM

[00126] We performed pseudotime analysis to understand the trajectory of our blood and immune progeny, which helped identify key genes with high rates of expression changes within each condition.

[00127] Figures 8A-K show aspects of gene expression over pseudotime and inferred cell-cell, cell-ECM, and cell-secretome interactions confirming myeloid matrix-remodeling phenotype in irradiated eBM cells. Expression values of key myeloid (Fig. 8A), matrix-associated (Fig. 8B), and MDSC genes (Fig. 8C) over pseudotime are shown. Fig. 8D shows comparative quantification and protein breakdown (Fig. 8E) of total inferred cell-cell, cell-ECM, and cell-secretome signals via CellChat algorithms. Figs. 8F-8K show inferred (Fig. 8F-H) secreted signals and cell-cell interactions (Fig. 8I-K) within each subpopulation over each experimental group. Figures 20A-C show inferred incoming and outgoing interactions within 1 Gy group via CellChat.

[00128] Interestingly, CD14 expression was consistent between groups, significantly increasing in the neutron group, and only recently in relative pseudotime were other canonical markers of M2-macrophagees (VCAN, ITGAM) and MDSCs (S100A8, S100A9) increasing in expression (Fig. 8A-C). Of note, genes relating to M2 macrophage phenotype and ECM remodeling, including CD 163, FN1, and MMP9, showed variable changes to expression levels over time, indicating that these cells were variably responding to systemic stimuli (Fig. 8A-B). We also used CellChat computational algorithms to predict the expected cellcell, cell-ECM, and secreted cell interactions in our subpopulations, showing an increase in interactions and interaction strength of cell-cell contacts and secreted signals in the 0 Gy Control group, as compared to the irradiated groups. The number of ECM interactions was much higher (709 molecules) in the neutron- irradiated group, but the interaction strengths of the photon- and neutron-sources were both larger than that of the 0 Gy Control group (Fig. 8D).

[00129] When comparing the neutron-irradiated samples with the 0 Gy Control, 32 distinct cell-cell contact proteins of interest were revealed, with significant differential expression of a subset of proteins, including APP, MPZ, GP1BA, NEGR, CD45, CADM, PVR, and NRXN, many of which are critical in leukocyte adhesion and motility (Fig. 8E). In neutron-irradiated secreted signals, significantly differentially expressed factors include IL16, ANNEXIN, SPP1, CXCL, IL2, APRIL, NRG, BAFF, and GDF (Fig. 8E). M2 macrophages, found to be present at high concentrations in these irradiated groups, also secrete high levels of SPP1, APRIL, and CXCL. In addition, high levels of secreted ANNEXIN have been shown to be indicators of cellular stress in tumor-associated macrophages. This increased M2 phenotype was reflected in higher inferred cell-ECM interactions with matrix proteins fibronectin, heparan sulfate proteoglycan (HSPG), and tenascin - all implicated in MMP9-induced cancer stem cell invasion (Fig. 8E). These predicted interactions can further be broken down into cell subtype, highlighting similarities between cell types (Fig. 8F-K). Figures 26A-I show additional aspects of the velocity analysis.

[00130] Unique biomarkers of neutron-mediated injury in eCT and. eBM tissues

[00131] To identify signatures of neutron-specific radiation injury, we compared significant and differentially expressed (p<0.05, L2FC>1) genes between eCT and eBM tissues, trying to elucidate differences caused by the photon and neutron doses.

[00132] Figures 9A-I show aspects of parallel analysis of photon and mixed neutron sources of radiation for eCT and eBM tissues. Fig. 9A shows top differentially expressed genes with highest absolute fold change in eCT tissues. Fig. 9B shows selected key hypertrophy-related genes are increased in neutron- irradiated eCT tissues. Fig. 9C shows GO biological processes pathways enriched in differentially expressed genes in eCT 4 Gy Photon versus 1 Gy Neutron comparative analysis. Fig. 9D shows the top differentially expressed genes with highest absolute fold change in eBM tissues. Fig. 9E shows the highest up- and down-regulated genes in the CD 14+ fraction of eBM-derived cells. Fig. 9F shows a comparative analysis of cell-cell interactions and secreted signals in 4 Gy Photon versus 1 Gy Neutron eBM tissues. Fig. 9G shows GO biological processes pathways enriched in differentially expressed genes in eBM 4 Gy Photon versus 1 Gy Neutron comparative analysis. Fig. 9H shows shared genes between control and neutron-irradiated tissues between eCM and eBM bulk analyses. Fig. 91 shows shared genes between photon- and neutron-irradiated tissues between eCM and eBM bulk analyses. *p<0.05,*** p<0.001, **** p<0.0001.

[00133] In eCT models, we observed an increase in known radiation-related genes, including PTGS2, L1F, 1CAM1, FOS, and OSG1N1 (Fig. 9A). We also found an increase in genes associated with cardiac hypertrophy (NPPB, MYH7, NEAT1 , FLNC, and CSRP3), and pathway analysis also revealed an increased presence of smooth muscle cell proliferation, CYP450 metabolism, and cellular responses to hypoxia and inflammatory stimuli (Figs. 9B-C; Figs. 21A-E).

[00134] Figures 21A-E show additional differentially expressed changes associated with eCM tissues and their response to 4 Gy Photon versus 1 Gy Neutron. Fig. 21A shows a principal component analysis of eCM tissues in response to either photon or neutron sources of radiation. Fig. 2 IB shows differentially expressed and significant genes between the two groups (p<0.05, log2foldchange>l ). Figs. 21C-E show individualized visualization of genes associated with cardiac function/maturation, cardioprotcction, and DNA repair.

[00135] In CD45 ⁺ cells isolated from eBM tissues, we were able to also find increases in genes relating to matrix remodeling (MMP1, MMP8, MMP12), chemotactic (CCL18, CCL8, CCL7, CCL2), collagens (COL1A2, COL1A1), and radiation (HES2, GAL3ST4, DDIT4, HRK), in response to neutron doses, as compared to photon-irradiated controls (Fig. 9D). Within CD14 ⁺ cells only, neutron doses seemed to increase the ECM-remodeling phenotype of the myeloid cells, with a downregulation in antigenpresentation genes HLA-DRA and CD74 (Fig. 9E). Using CellChat and secreted signal analysis, TWEAK appeared to be significantly secreted by neutron-irradiated cells, indicating a role in the TNF-stimulated apoptosis (Fig. 9F). Differential gene expression revealed similar changes in ECM remodeling in response to neutron rays, potentially giving insight into a potential neutron-specific affect in macrophage remodeling capacity (Fig. 9G).

[00136] We were also interested in understanding whether there were neutron-specific changes that could be observed amongst multiple human tissues, in our case amongst hematopoietic cells and cardiac muscle. Within the significant differentially expressed genes overlapping between eCT and eBM data, we observed 25 genes of interest responding to radiation and neutron-induced damage, including known genes associated with tumor suppressor p53, including MIR34AHG and PHLDA3. We also noted a potential implication in a pro-fibrotic and senescence-related radiation injury, which could be suggested by COL24A1. When comparing the neutron-irradiated samples to photon groups, hoping to isolate the effect of just high-LET radiation sources, we identified 12 common genes between eCT and eBM models, including a number of genes related to oxidative stress, including HM0X1, HRK, and MIR22HG.

[00137] Radioprotective drugs mitigated radiation damage in eCT and eBM tissues

[00138] To demonstrate the utility of our platform for future studies of radioprotective agents, we observed the functional changes to each tissue platform in response to either Amifostine, a pre-treatment FDA-approved radioprotective drug, and granulocyte-colony stimulating factor (G-CSF), an FDA- approved radioprotective cytokine that stimulates marrow recovery post-radiation in vivo.

[00139] Figures 22A-M show aspects of using engineered human tissues for therapeutic testing of radioprotective agents. Figures 22A-J shows that pre-treatment of eCM tissues with radioprotective drug Amifostine prior to acute exposures prevented the abnormal hypertrophic-like functionality of neutron-irradiated tissues. Figures 22K-L shows that G-CSF stimulation increases CD45+ cell production by increasing neutrophil production in vitro (Fig. 22M).

[00140] eCT tissues exposed to neutron rays but pre-treated with 1 mM Amifostine demonstrated remarkable return of functional metrics to those of healthy controls (Figs. 22A-J). Notably, the excitation threshold and maximum beating frequency were maintained at control levels with drug administration. The only metric that was significantly different between the neutron-and-drug-treated group and the control group was in the time of muscle relaxation, which was still not at the relaxation period of the healthy control. In the eBM, G-CSF treatment was administered at a low total dose (5 ng/mL) for 8 days post-radiation, which helped increase production of CD45+ blood cells (Fig. 22K-L) through increases in neutrophils and subsequent proliferation of multipotent progenitors in vivo (Fig. 22M). This proof-of-concept experiment helped provide some insights into the potential mitigation of radiation-induced tissue changes.

[00141] We also investigated a responsive bone marrow niche for studying tissue cryogenic injury and repair in a multi -tissue system. In the human body, the bone marrow is the site of blood and immune cell production and is responsible for production of immune cells to respond to injury and disease. In the heart, specifically after myocardial infarction or other cardiac injuries, micro-RNAs and other signaling molecules travel from infarcted hearts to the bone marrow via exosomes. The exosomes downregulate CXCR4 signaling on hematopoietic progenitors, causing an increased egress of blood progeny into circulation and increased homing to the site of injury. To mimic this phenomenon with our primary CB-BM and iBM tissue models, we integrated this model system with engineered cardiac muscle tissues (eCTs) using our previously established multi -organ-on-a-chip system (multi-OOC). The platform we used is able to separate an individual tissue compartment from the circulatory perfusion compartment via an endothelialized barrier, allowing for selective cell and secreted factor cross-talk between multiple engineered tissue models. Once integrated, we attempted to demonstrate cryoinjury to the cardiac muscle by exposing the engineered cardiac tissue to temperatures below -40 °C. or below -50 °C., such as -79 °C., which in turn stimulated immune cell migration from the eBM variations through the vascular circulation compartment and into the cardiac muscle in a multi -organ chip setting.

[00142] To use this model, we first engineered our eBM and eCT tissue models in isolation over 4+ weeks, then integrated them within the tissue platform for systemic studies. In this experiment, we wanted to model preferential homing of disseminated immune cells to a site of injury, so we integrated a BM module (either the CB-BM or iBM tissue model) in combination with 1 healthy (uninjured) eCT and 1 cryoinjured eCT model.

[00143] Figures 23A-D show aspects of recruitment of eBM-derived blood and immune cells in a multiorgan chip system. Fig. 23 A shows a schematic overview of the experimental work. Fig. 23B shows evidence of cardiac-specific troponin release in the cardiac muscle compartment at D10 post-injury. Fig. 23C shows flow cytometric characterization of immune cells recruited into the cardiac muscle tissues at Day 10 from integrated cultures with CB-BM tissues. Fig. 23D shows flow cytometric characterization of immune cells recruited into the cardiac muscle tissues at Day 10 from integrated cultures with iBM tissues. *p<0.05; **p<0.01; ***p<0.005 with paired student’s t-tests.

[00144] Ten days after tissue injury, we characterized the cells in each compartment and the eCT injury itself to ensure we were able to mimic tissue-level damage via cardiac troponin secretion and cytokine release.

[00145] Figures 24A-I show aspects of eBM-released CD45+ blood cells in circulation and on endothelial barriers 10 days post-injury. Fig. 24A shows a schematic overview of the cells analyzed in Fig. 24B. Fig. 24B shows flow cytometric characterization of CB-BM and iBM-derived cells in circulation. Fig. 24C shows a schematic overview of the cells analyzed in Fig. 24D. Figs. 24D-I shows flow cytometric characterization of CB-BM and iBM-derived cells attached to endothelial barriers. *p<0.05; **p<0.01; ***p<0.005 with Student’s T-Tests (A) or Two-Way ANOVAs (B).

[00146] We characterized the ability of the vascular barrier to permit release of CD45+ and CDl lb+ blood cells into the circulation compartment in both CB-BM and iBM tissue formats (Fig. 24A-D). Similarly, we noticed that these CD45+ blood cells were present at highest quantities on the bone marrow barrier, which is expected as the site of CD45+ cell production and release into the systemic circulation (Figs. 24D-I). Interestingly, there were no significant differences between the percentage of blood cells found on the endothelial barriers of the control versus injured eCTs (Figs. 24D-I).

[00147] Once cells were dissociated from the control and injured eCTs themselves, there were increased percentages of CD45+, CDl lb+, CD14+, and CDl lc+ DCs in the injured tissues as compared to the controls (Fig. 23A-D). This effect was most significant between eCTs in integrated culture with CB-BM tissues (Fig. 23C), though many of the eCTs in culture with iBM tissues were also significant (Fig. 23D).

[00148] Figures 25A-L show aspects of cytokine responses to cryoinjury. Figs. 25A-D shows differential expression levels of key inflammatory cytokines IL-6 and MCP-1 in cardiac muscle tissue compartments in either CB-BM or iBM integrated cultures. Figs. 25E-L shows cytokine concentrations in circulation for selected inflammatory cytokines at Days 3 and 10 for CB-BM (grey) or iBM (blue) integrated cultures.

[00149] We demonstrated herein that engineered human tissue models can be used to study the effects of high energy radiation designed to mimic that encountered during long-range space travel such as the Mars mission. By using a combination of primary cells and iPSCs, we were able to recapitulate organ level responses of human bone marrow and heart to radiation, including the generation of downstream blood and immune progeny in the cBM and contractile behavior of cCT. These human tissue models captured the functional and molecular changes associated with radiation injury, including the emergence of unique myeloid populations following neutron radiation exposure, and differential expression of key genes in both the eBM and eCT following neutron vs photon radiation exposure. These shared genes associated with neutron radiation exposures are key in identifying unique biomarkers of secondary radiation damage in deep space missions (i.e., MIR22HG, HM0X1), as neutrons are among the greatest risks for astronauts on spacecrafts headed to Mars. We believe that our study is the first to use engineered human tissue models for studying simulated galactic cosmic rays, as well as establish a proof-of-concept platform and framework for using engineered human tissue models for mitigating other NASA “Red Risks” on Earth.

[00150] In the eCT studies, neutron-irradiated tissues had impaired conduction and excitability, as evidenced by the decreased maximum capture rate and increased excitation threshold, indicating that more stimulation would be necessary to excite the irradiated eCT tissues. However, these irradiated eCT tissues also demonstrated remarkably higher force generation and contractility to their control counterparts, which may indicate an early hypertrophic phenotype of the neutron-irradiated tissues. This increased force generation was corroborated by the increase in cardiac-specific genes and ontology pathways, as well as increases in hypertrophy-related genes MYH7, NPPB, and NEAT1. Notably, HM0X1 was significantly upregulated in neutron-irradiated muscle compared to not only the untreated controls but also to the photoirradiated eCT tissues, indicating that the neutron-specific dose was critical in activating the stress-response pathway normally implicated in oxidative stress or hypoxic injury to the myocardium. Other groups have reported increases in oxidative stress of cardiomyocytes in response to radiation, and increases in cardiacspecific gene expression levels in response to high (>20 Gy) photon doses. Many studies of radiation- induced cardiac dysfunction were focused on microvascular changes in the heart. Although there was no endothelial compartment in our model, neutron-irradiated eCT tissues exhibited downregulated ontology pathways associated with vascular maturation, potentially indicating a repressed role of angiogenic factors in the irradiated eCT tissues.

[00151] We observed the expected responses of the radiosensitive hematopoietic system to neutron doses, most notably the decreased proliferation of CD45+ cells and increased inflammatory signatures at early time points, similar to cell monolayers and animal studies of similar doses. The reduction of the hematopoietic lymphoid compartment and skewing towards myeloid lineages are unique aspects of the models described here, in line with the available data from human studies, in nuclear bystander patients in atomic bomb survivors, and in non-human primate studies.

[00152] Human data from recent spaceflight studies indicate a skewing of hematopoiesis into myeloid progenitors and increased development of pre-malignant myelogenous clonal hematopoiesis. Also, data from ex vivo irradiation of peripheral blood indicates a decrease in the lymphoid compartment in response to neutrons over the course of a few days. These previous studies, in tandem with our data, may indicate the role of high-LET exposure in promoting a pre-malignant phenotype in HSPCs, skewed towards a myeloid-biased output and eventual progression into an AML-like manifestation. Unique myeloid populations emerging in our system three weeks post-irradiation include M2-macrophage-like and MDSC- like cells, indicating a reparative role in preventing further tissue inflammation and promoting matrix remodeling, both phenotypes seen in pre-malignant and malignant tissues. However, more work is needed to better understand the phenotype of these pre-malignant cells, and whether these cells can restore normal hematopoiesis when the irradiated stimuli are resolved post irradiation.

[00153] As the fields of stem cell biology and OOC continue to develop, more technologies are emerging to address the need for human-relevant models for studies of radiation on human health. Animal models and cell monolayer studies are the gold-standards for studying the differential effects of neutron sources as compared to photon and GCRsim studies, though there is little work specifically targeting human cells - and no work, to date, on human tissues. Because the RBE of neutron exposures is higher than in photon rays, and varies greatly between organ types in the body, developing individual, organ-specific human tissue models is of great interest to the scientific community. Previous work by other groups has shown the utility of human OOC models at mimicking the acute radiosensitivity of the gut and marrow in response to low levels of gamma radiation. However, these studies are limited to short-term (hours to days) responses to radiation, showing immediate cell death of proliferative cells in response to high doses of gamma rays. Studies with isolated model systems, like those we present here, allow for the unique ability to parse out individual factors contributing to radiotoxicity or radioprotection in space-relevant radiation sources. OOC models may address the needs of studying the differences between acute and protracted doses, as the dose rate and delivery frequency of exposures varies greatly in deep space as compared to radiotherapy strategies used in the clinic.

[00154] OOC models may allow for studies of other NASA “Red Risks,” both in isolation and in tandem with radiation, including studies of hypoxia or microgravity. Personalizing these model systems may allow for unprecedented understanding of how an astronaut’s individual organ health may be impacted by space travel. It is critical, however, to compare data obtained in humanized radiation studies on Earth with those of past nuclear exposures (i.e., Hiroshima, Chernobyl) and clinical data animal studies. More recently, human-specific data has emerged from short-term studies of human cells in LEO experiments on the ISS, as well as data collected from astronauts (i.e., NASA Twins Study) after their return to Earth.

[00155] Our results show both functional and molecular changes associated with human OOC models of the bone marrow and heart after exposures to either photons or neutron radiation. In preparation for a future Mars mission, and on Earth, in the unexpected incidence of an accidental nuclear exposure, this model can be used to develop new radioprotective countermeasures to circumvent downstream damage on human health in response to damaging neutron rays or other high LET radiation. Some radioprotective agents have been studied over the past 50+ years to prevent downstream damage to the body, but none of these drugs were successful in preventing phenotypic changes in all organs. We show in a “proof-of-concept” experiment that exposure to radioprotective drugs Amifostine (pre-treatment of the eCT) and G-CSF (posttreatment of the eBM) was able to ameliorate some of the neutron-associated changes. Although these drugs may provide some alleviation of injury , in long-range Mars missions with radiation from multiple ion sources and delivered over a multi-month-long journey, new therapeutics need to be developed to provide protection of all sensitive organs, such as the hematopoietic and cardiovascular systems. OoCs provide an ideal platform to help validate new therapeutic modalities, as those that work in animals in response to radiation may not work in humanized platforms and by extension to humans.

[00156] We studied the effect of radiation on a hematopoietic system without significant exogenous stimuli (i.e. cytokines), which allows for studying hematopoietic differentiation into downstream progeny, rather than maintenance of the HSPC phenotype in particular. Uniquely, this approach allowed for careful characterization of the downstream populations emerging in response to radiation.

[00157] Using human tissue models, we found the biological effects of neutrons to be strongerthan those of photons, and to vary greatly between endpoints and the two organ types studied. Because of the differences in responses to radiation from one individual to another, developing individualized, organspecific human tissue models is of great interest to the space-science community. Previous work has shown utility of human OoC models for mimicking the acute radiosensitivity of the gut and marrow in response to low levels of gamma radiation. Most recently, Verma et al. studied the role of astrocytes in simulated GCR injury for up a week using a blood-brain barrier OoC model. However, these studies are limited to short-term (hours to days) responses to radiation, showing immediate loss of viability of the most proliferative cells in response to radiation injury. Studies with tissue model systems that we present here allow uniquely to parse out the individual factors contributing to radiotoxicity and radioprotection for radiation sources and dosages relevant to space travel.

[00158] We studied the responses of the cardiac and bone marrow tissues to neutron radiation over three weeks post-radiation, a period providing just a snapshot of the time-dependent effects of acute radiation exposure on human tissues. In future studies, these changes should be studied over longer periods of time and extended to the cellular repair mechanisms. To this end, we are currently extending the lifetime of OoC platforms to six months or longer.

[00159] Future studies would greatly benefit from increased throughput and evaluation of individualized responses to radiation. Biological variables that can influence an individual’s response to radiation, including sex, race, and genetic background. The model systems described here can be personalized, by developing an entire tissue model for the same individual using iPSCs.

[00160] Future work could also address the functional changes to HSPCs in the early (hours/days) and late (weeks/months) periods post radiation. Because we observed a shift in phenotype towards a myeloid- biased hematopoietic system, as seen in clinical exposures of radiotherapy and astronauts returning to Earth, we can further characterize the effects of these acute doses on the emergence of myeloid pre/malignancies. Mutational analysis of irradiated HSPCs may help identify genetic and epigenetic-level changes leading to the development of clonal hematopoiesis, as shown in the NASA Twins Study.

[00161] We describe herein the integration of an eBM tissue model with either healthy (uninjured) or cryoinjured cardiac muscle tissues (eCTs), for studies of acute systemic insult to one tissue compartment. The goal of these studies was to elucidate whether our engineered, multi-organ tissue model would be able to mimic the systemic changes to the body when one or more organs are subjected to an acute insult, causing recruitment of cells from the BM to aid in inflammatory responses and remodeling of the injury site. To our knowledge, this is the first report demonstrating a human-specific model to mimic heart injury via mobilized BM progenitors/progeny.

[00162] In many multi -organ studies to date, investigators have supplemented their cultures with either primary or cell line-based immune cells in circulation to mimic and respond to inflammatory events. While this is effective at studying a number of acute injuries and diseases with a generalized immune cell population (i.e., monocytes), this does not closely mimic the responsiveness of the bone marrow to emergency stimuli, and in turn, suppresses the variety of blood and immune cells that are responsible for early- and late-stage injury states. Further, mimicking the constant production of blood and immune cells in the body allows for recapitulation of the body during multiple cycles of the wound healing process, as well as in cases where these insults to off-target tissues can last for weeks to months.

[00163] In this work, we demonstrate a “proof-of-concept” model of acute cardiac injury with recruitment of cells from a human bone marrow compartment, mimicking closely the infiltration of immune cells during an injury to the cardiac muscle, as in ischemia-reperfusion injury or drug-induced cardiotoxicity. This model system, however, is not limited to injuries to the cardiac muscle, and in future work, can be applied towards studying any number of acute and chronic injuries to off-target organs. In much of our ongoing work in the group, we are attempting to characterize the effects of acute infection, as in COVID-19, on recruitment of cells to the site of viral invasion (i.e., uptake of viral particles in the lungs) as well as sites of secondary infection (i.e., myocarditis in the heart). We believe complex, multi-organ platforms like this can allow for study of systemic immune-mediated injury states that are difficult to recapitulate with singleorgan models.

[00164] Finally, linking the engineered human tissues into a multi-organ context allows for studies of systemic injury such as due to radiation toxicity or cryoinjury, showing cross-talk between human organs to mimic the most realistic effects of injury and recovery. Extension to a “patient-on-a-chip” model could provide a personalized platform for optimizing protection from the harmful effects of radiation. [00165] Although the data presented herein relate primarily to changes in tissues due to irradiation or cryoinjury, other diseases can be studied using similar models.

[00166] We describe a bioengineered bone marrow system to study a variety of human diseases, toxicity responses, and immune functions in vitro. Derived from multiple cell sources, we have engineered an all induced pluripotent stem cell (iPSC) bone marrow model comprised of osteoblasts, mesenchymal stem/stromal cells, endothelial cells, and hematopoietic stem and progenitor cells (HSPCs). Using decellularized bone scaffolds as a backbone, iPSC-derived component cells are encapsulated in fibrin or matrix-derived hydrogels in combination, and we can study the progression, proliferation, and differentiation of hematopoietic cells in an in vitro setting. In addition to iPSC-derived sources, we are able to create the same bone marrow model using primary human cell sources (i.e. primary MSCs, endothelial cells including HUVECs, and cord blood- and bone marrow-derived CD34+ HSPCs). We have also shown the model is able to respond to external cues, like drug treatment, cytokine cues, and/or radiation damage.

[00167] This is the first, to our knowledge, model of the bone marrow to incorporate entirely iPSC-derived stromal cells (osteoblasts, endothelial cells, and mesenchymal stem/stromal cells), as well as the first 3D, nichelike microtissue environment for the support of iPSC-derived HSPCs. Further, our model has components, in a spatially organized fashion, of the endosteum (osteoblasts, MSCs) and perivascular space (MSCs, endothelium) for controlled distribution of blood stem cells and their progeny. Our engineered bone marrow model can fill the role of a patient-specific, controllable, and complex bone marrow niche to support HSPCs in vitro and allow for modeling of genetic blood/immune disorders for up to 28 days, or more. For certain variations of the model, we have cultured the tissues for up to 12 weeks in total culture time.

[00168] Further, we demonstrate the use of the entirely iPSC-derived and primar -derived bone marrow microtissue models for studying healthy hematopoietic stem cell proliferation/differentiation, radiation toxicity, and blood cancers.

[00169] We believe our platform helps solve the critical problem that animal models poorly recapitulate human physiology, especially in human blood cancers. Our platform is the first, to our knowledge, to include iPSC-derived stromal cells of the marrow into a 3D microtissue, containing healthy hematopoietic cells from primary (cord blood or bone marrow-derived CD34+ cells) or iPSC-derived sources (CD34+). We demonstrate ability to maintain progenitors (CD34+) and promote differentiation of cells into downstream lineages (myeloid, lymphoid-lineage committed cells).

[00170] The ability to study both healthy and malignant blood cells (and their microenvironmental factors) is critical to understanding the progression of disease and treatment mechanisms (i.e., radiation, chemotherapy). [00171] Our model is an engineered tissue platform, allowing for tissue architecture of the bone to be recapitulated, as well as ability to derive extracellular matrix components in a spatially organized architecture. [00172] Our model allows for modularity of cell types, so different cell derivations can be combined to allow for cell type-specific studies. For example, if you genetically alter one cell type (i.e. MSCs), you can study the role of that specific cell with a knock down or knock in of a specific gene in conjunction with an otherwise healthy system. In addition, we can incorporate healthy and malignant hematopoietic cells from the same donor within the same model,.

[00173] Our model includes cells from either all iPS cell sources or primary cells, or a mix.

[00174] Our model describes applications in studying cosmic radiation in engineered human bone marrow from isogenic cell sources. It is capable of being incorporated into systems for multi-organ interactions and scalable. The model can be used to study viral injury and response, multi-organ injury and recruitment of immune cells from a bone marrow compartment, and bone marrow's changes during cancer metastasis.

MATERIALS AND METHODS

[00175] Study design: Our objective was to engineer multiple multicellular tissue models, capable of recapitulating key features of each target organ (i .e. bone marrow, cardiac muscle), in response to radiation injury. We were interested to understand the longer-term (weeks) changes acute doses of radiation may have on human tissues, mimicking doses that will have large enough doses to affect tissue function and physiology. In addition, we chose both photon radiation (involved in cancer therapy) and neutron sources (secondary rays from cosmic radiation), to mimic the potential changes associated with human tissues on a mission to deep space. We chose the two tissue models of interest to represent a tissue known to be radiosensitive (bone marrow), with acute changes to hematopoietic cells, and a tissue affected by chronic changes (cardiac muscle), with downstream changes to function. Further, we expected to identify potential genes altered in radiation exposures, particularly those that are maintained three weeks post-radiation in our in vitro culture platforms, as well as those that may be shared amongst the two target organs (bone marrow and cardiac muscle). In addition, we were interested in neutron-specific changes associated with the type of radiation rather than the dose, as neutrons are approximately four times as potent as photon sources.

[00176] Radiation dosing: Photon irradiations were performed at Columbia University’s Center for Radiological Research using a Gammacell 40 ¹³⁷ Cs irradiator (Atomic Energy of Canada Ltd) with a dose rate of 0.68 Gy/min for varying low-energy doses (i.e. 1, 2, 4, 6 Gy). Neutron irradiations were performed at the Columbia University Radiological Research Accelerator Facility (RARAF), using an accelerator- based neutron irradiator mimicking the neutron energy spectrum from an Improvised Nuclear Device. Briefly, a mixed beam of atomic and molecular ions of hydrogen and deuterium is accelerated to 5 MeV and used to bombard a thick beryllium target. The energy spectrum of neutrons emitted at 60° to the ion beam axis closely mimics the Hiroshima spectrum at 1-1.5 km from the epicenter. During irradiation, samples were placed below and in front of the beryllium target, at an angle of 60° to the particle beam and a distance of 17.5 cm. Irradiations were performed with a total beam current of 20-30 pA, resulting in a dose rate of 2-3 Gy/h of neutrons with an additional 20% of concomitant y rays, for varying high-energy doses of (i.e., 1, 2, 4, and 6 Gy). Dosimetry was performed at the beginning of each irradiation day, using a custom built Tissue Equivalent Proportional Counter (TEPC)(62), which measures total dose and a compensated Geiger-Mueller dosimeter, which has a very low response to neutrons, and thus measures only the photon component.

[00177] Cardiomyocyte differentiation from human iPSCs: hiPSCs (WTCl l-GCaMP6f line was obtained through material transfer agreements from B. Conklin, Gladstone Institutes. Cardiomyocytes were differentiated as previously described (28). On Day 10, RPMI-no glucose (Life Technologies, 11879020) supplemented with B27 (Thermo Fisher Scientific, 17504044) and 213 pg/mL ascorbic acid (Sigma- Aldrich, A445), was used to purify the iPSC-CMs population and eliminate any contaminating mesodermal and endodermal populations. Medium was replaced on day 13 with RPMI-B27 supplemented with 213 pg/ml ascorbic acid until day 16. On day 17 cells were pretreated with rock-inhibitor (y-27632 dihydrochloride, 5 pM) for 4 hours before dissociation. Cells were dissociated by enzyme digestion with collagenase type II (95 U/mL; Worthington, LS004176) and pancreatin (0.6 mg/mL; Sigma-Aldrich, P7545) in dissociation buffer (Glucose (5.5 mM), CaC12-2H20 (1.8 mM), KC1 (5.36 mM), MgSO4-7H20 (0.81 mM), NaCl (0.1 M), NaHCO3 (0.44 mM), NaH2PO4 (0.9 mM)) on a shaker in a 37°C incubator. Flow cytometry for cTnT+ (BD BioSciences, 565744) was performed prior to cell use fortissue fabrication to ensure cell purity (>90% cTnT+).

[00178] Engineering of cardiac tissues: We generated eCTs using our previously published “milliPillar” cardiac microtissue platform. Briefly, primary human ventricular cardiac fibroblasts (NHCFV; Lonza, CC- 2904) were cultured according to the manufacturer's recommendation. Differentiated hiPS-CMs were dissociated and mixed with supporting cardiac fibroblasts in a 75:25 (hiPS-CM:NHCF-V) ratio. The cells were subsequently resuspended in fibrinogen by mixing the cell solution with 33 mg/mL stock human fibrinogen (Sigma- Aldrich, F3879) and RPMI-B27 (RPMI 1640 basal medium, L-ascorbic acid 2- phosphate, and B27 supplement) to a final fibrinogen concentration of 6.25 mg/mL and a cell concentration of 45,833 cells/pL. 3 pL of thrombin solution (2U/mL) were added to each well, followed by 12 pL of the cell/fibrinogen solution. The solutions were mixed and allowed to polymerize at 37 °C for 15 min, so that the tissues readily formed around the pillars. These resulting tissues each contained 550,000 cells in a 5 mg/ml fibrin hydrogel.

[00179] 400 uL of RPMI-B27 with lOuM Rock inhibitor and 5 mg/mL 6-aminocaproic acid (Sigma- Aldrich, A7824) were added to each well. After 24 hours, the medium was changed to RPMI-B27 with 5 mg/mL 6-aminocaproic acid and replaced every other day. On the fifth day following tissue formation, the medium was changed to RPM1-B27 without 6-aminocaproic acid and replaced every other day. On the seventh day following tissue formation, electrical stimulation was initiated to promote tissue maturation. Tissues were stimulated at 2 Hz with a 5 V/cm electric field provided by 2 ms biphasic pulses throughout culture period.

[00180] Tissues administered with Amifostine trihydrate (generously donated by Clinigen and Zambon Chemicals; 112901-68-5) were incubated with 1 mM Amifostine for 30 minutes at 37 DC prior to irradiation, with all tissues receiving fresh media prior to irradiation.

[00181] Generation of engineered cardiac muscle tissue for multi-tissue study: Briefly, hiPSC- derived cardiomyocytes (WTC-11) were differentiated using previously described chemically defined canonical Wnt stimulation and inhibition protocol. Day 10 cardiomyocytcs were proliferated until passage 3 using a previously described cardiomyocyte expansion protocol. A ratio of 75% hiPSC-derived cardiomyocytes were combined with 20% human ventricular cardiac fibroblasts (NHCF-V; Lonza, CC- 2904) in 84 pl 3 mg ml-l human fibrinogen (Sigma, F3879) and cross-linked with 16 pl 100 U ml-l thrombin from human plasma (Sigma, T6884) to form a hydrogel between two flexible pillars. After 20 min of cross-linking at 37 °C in 5% CO, cardiac medium was added (RPMI 1640; ThermoFisher, 11875-093; B-27 supplement (serum free, ThermoFisher, 17504044); and penicillin/streptomycin (Gibco, 15070063)) supplemented with 3mg ml-1 6-aminocaproic acid (Sigma, A2504). After 1 week of compaction, heart tissues were subjected to electromechanical conditioning at a frequency increasing from 2 to 6 Hz.

[00182] Derivation of bone scaffolds: Bovine calf metacarpals were purchased in bulk and stored at — 40°C (Lampire Biological Laboratories, #19D24003). A band saw is used to obtain a section (~4 cm tall) from the distal end of the metacarpal, as this region contains an enriched concentration of trabecular bone. We then used a CNC Milling machine to acquire smaller rectangular bone cores with a cross section of 4 mm x 8 mm. These pieces were then placed into an IsoMet low speed wafering saw to then cut each individual piece into 1 mm thick bone scaffolds. Each scaffold was approximately 4 mm (width) x 8 mm (height) x 1 mm (depth), and subsequently processed for removal of cell debris.

[00183] Decellularization protocols were adapted from our previously established protocols, which removed all cellular material but preserved the matrix composition and architecture of the bone. Bone scaffolds were processed in batch with the following step-wise protocol all completed on an orbital shaker: (i) PBS with 0.1% EDTA (w/v) for 1 hour at room temperature; (ii) 10 mM tris, 0.1% EDTA (w/v) in DI water overnight at 4°C; (iii) 10 mM Tris, 0.5% sodium dodecyl sulfate (w/v) in DI water for 24 hours at room temperature; (iv) 100 U/ml DNase, 1 U/ml RNase, 10 mM Tris in DI water for 6 hours at 37°C. After decellularization was complete, bone scaffolds were lyophilized until freeze-dried using a Labconco freezone lyophilizer (7740020), and cut to a final scaffold size of 4 mm x 4 mm x 1 mm. Scaffolds were weighed to ensure each piece was at the appropriate density for cell seeding (5-7 mg per scaffold), and cut in half to reach a final scaffold. For sterilization, bone scaffolds were subjected to 70% ethanol treatment overnight, and then washed with DMEM basal media overnight.

[00184] Engineering of bone niche and bone marrow tissues: Induced pluripotent stem cells (iPSCs) were expanded on Matrigel-coated plates (Coming) for 2 weeks prior to differentiation. WTC-1 1 iPSCs were differentiated using the STEMdiff™ Mesenchymal Progenitor Kit according to manufacturer’s instructions into iPSC-dcrivcd MSCs (iMSCs) over a period of three weeks (Stem Cell Technologies, 05240). iMSCs were expanded and seeded into the bone scaffolds at a concentration of 2 x 10 ⁵ cells per scaffold, using 15 pL of medium (4.5 g/L DMEM supplemented with 10% (v/v) HyClone FBS, 1% penicillin/streptomycin, and 1 ng/mL of basic fibroblast growth factor, bFGF), according to established protocols. The cells were allowed to attach for 2 hours, and then supplemented with additional MSC medium overnight. After 72 hours, osteogenic differentiation of the seeded cells was initiated with the addition of low glucose (1 g/L) DMEM supplemented with 1 pm dexamethasone (Sigma Aldrich), 10 mm 0-glycerophosphate (Sigma Aldrich), and 50 pM L-ascorbic acid-2 -phosphate (Sigma Aldrich). Each scaffold was incubated in 1 mL of osteogenic media, with media changes 3 times a week for 4 weeks, allowing for the iMSCs to differentiate into functional, maturing osteoblasts.

[00185] Following osteogenic differentiation, human umbilical vein endothelial cells (HUVECs; Lonza, C2519A) and additional iMSCs were added within a 10 uL fibrin hydrogel (11 mg/mL fibrinogen, Sigma Aldrich, F3879; 33 U/mL thrombin, Sigma Aldrich, T6884) to each scaffold.

[00186] Microtissues were then incubated in endothelial cell growth medium -2 (EGM-2; Lonza, CC- 3162) for 1 week with addition of 33 mg/mL of the protease inhibitor aprotinin (Sigma-Aldrich, A3428) and placed on a rocker (Cole Parmer; 51401-00). After 1 week, media was removed from the microtissues and 1.1 x 104 cells/tissue of mixed donor cord blood (CB)-derived, CD34+ human HSPCs (Stem Cell Technologies, 70008) were allowed to attach for 2 hours at 37°C in a humidified incubator at 5% CO2. After seeding, tissues were replenished with StemSpan™ SFEM II medium with 1% P/S (Stem Cell Technologies, 09655) with 10 ng/mL stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT-3L) (Peprotech) for the first four days (prior to radiation exposure). After Day 4, tissues underwent half media changes every four days with 5 ng/mL SCF, TPO, FLT-3L in SFEM II medium. Microtissues were kept in culture for 21 days post-irradiation. Tissues administered with recombinant human Granulocyte colony stimulating factor (G-CSF) (Peprotech; 300-23) were treated with 5 ng/mL G-CSF for 8 days and analyzed on Day 12.

[00187] Cardiac calcium imaging and analysis: To visualize calcium handling in real-time, WTC11- GCaMP6f iPSCs that contain a constitutively expressed GCaMP6f calcium-responsive fluorescent protein inserted into a single allele of the AAVS1 safe harbor locus were used (65). Tissues were imaged in a livecell chamber (STX Temp & CO2 Stage Top Incubator, Tokai Hit, Fujinomiya, Japan) using a sCMOS camera (Zyla 4.2, Andor Technology) connected to an inverted fluorescence microscope with a standard GFP filter set (Olympus IX-81). Tissues were electrically stimulated, and videos were acquired at 20 frames to measure tissue excitability and calcium flux as previously described. Calcium signals were analyzed from calcium imaging videos as previously described. Briefly, a custom Python script was developed to average the pixel intensities for each frame. This transient was then corrected for fluorescent decay. The SDRR, Tau, FWHM, FW90M, Contract 90, Contract 50, Relax 50, and the Relax 90 were calculated for every transient.

[00188] Cardiac brightfield imaging and analysis: For force generation measurements, videos were acquired at 20 fps using a custom program to stimulate cardiac tissues as previously described and force generation was analyzed from brightfield videos (28). Briefly, a custom Python script was developed to track the motion of the pillar heads and to calculate the force by multiplying the displacement of the pillars with the coefficient determined from the force-displacement calibration curve generated for the pillars.

[00189] Cardiac histology: Tissues were fixed in 4% paraformeldehyde, washed 3 times in IX PBS, and either kept for whole-mount imaging in PBS or processed for histological sectioning with paraffin embedding (Columbia HICCC Pathology core). Whole mount imaging was performed with primary antibodies for anti-a-actinin antibody (Sigma; A7811), anti-cardiac troponin T (ThermoFisher; MS-295- Pl), or anti-COLlAl (Abeam; ab34710), and mounted in CoverWell™ Imaging Chambers (Grace BioLabs; 631021).

[00190] eBM flow cytometry: the extent of □-H2AX in irradiated cells from eBM was characterized using the Amnis Image Stream Mk II cytometer to characterize the intensity and number of -H2AX foci per cell. 1-hour post-exposure to radiation, cells were fixed with BD Cytofix/Cytoperm according to manufacturer’s instructions and stored at 4°C. When staining, cells were incubated with PE anti -human H2AX antibody (Milteyni), FITC anti-human CD45 (BioLegend), Alexa Fluor 647 anti-human CD34 (BioLegend), and Hoechst 33342 (ThermoFisher) at room temperature for 1 hour and then washed with IX PBS three times Images of 1 ,000-1 ,500 cells per group were acquired at 60X with extended depth of field (EDF) for clearer visualization and identification of the fluorescent nuclei foci. Compensation coefficients between wavelengths of light were calculated using the compensation wizard in the Image Data Exploration and Analysis Software (IDEAS). After imaging, the spot wizard in IDEAS was used to quantify total foci per cell, total and mean fluorescence intensity each focus, area of foci, and area of nucleus.

[00191] eBM flow cytometry: other analyses of cells from the eBM tissues. Cells were collected in FACS Buffer (2% FBS, 0.5 mM EDTA in PBS), washed, blocked with human Fc blocker solution (Milteyni; 130-059-901), and prepared for flow cytometry with the following antibodies: BV421 antihuman CD45 (Biolcgcnd; 368522), APC anti-human CD34 (BioLcgcnd; 343608), BV605 anti-human CD38 (BioLegend; 356642), BUV395 anti-human CD90 (BD Biosciences), PE antihuman CD45RA (BioLegend; 304108), propidium iodide (Invitrogen/ThermoFisher; P3566), CD 14 (BV605), APC antihuman CD15 (BioLegend; 125617), BUV395 CDl lc (BD Biosciences; 563787), PE anti-human CD16 (BioLegend; 360704), and BV421 anti-human CDl lb (BioLegend; 301324). Flow cytometry was performed on a BioRad ZE5 machine and analyzed with FlowJo (BD Biosciences).

[00192] Colony forming unit assay: Cells were collected from the suspension compartment of eBM, counted, and plated in MethoCult™ SF H4636 (Stem Cell Technologies) in SmartDish™ 6-well, meniscus- free plates (Stem Cell Technologies) and placed within humidified chamber dishes (245 mm x 245 mm Square Dishes; Stem Cell Technologies; 27141). Plates were imaged on Day 14 using a BioTek Cytation 5 automated live-cell fluorescence imaging system with BioSpa automatic, incubated chamber (Columbia HICCC Confocal and Specialized Microscopy Core). Colonies were blindly and manually analyzed from images.

[00193] Histology: eBM tissues were fixed in 4% paraformaldehyde overnight at 4°C, washed three times with IX PBS, decalcified with Osteosoft (EMD Millipore; 101728) overnight at room temperature (only bone marrow tissues), washed three times with IX PBS, and paraffin-embedded for histological sectioning. All tissues were processed for hematoxylin and eosin (H&E), trichrome, or pentachrome by the Columbia University HICCC Molecular Pathology Lab. Paraffin-embedded tissue blanks were hydrated, processed for antigen-retrieval using a 10 mM sodium citrate buffer for 20 min in heat, and permeabilized with 0.25% (v/v) Triton-X for 20 minutes. Samples were then blocked for 2 hours with 10% FBS, and individual staining protocols followed for different tissues. Tissues were stained for immunofluorescence with Rabbit anti-human Bone sialoprotein antibody (ThermoFisher; PA5-79424), anti-Human/Mouse CXCL12/SDF-1 antibody (R&D Systems; MAB350), and Rabbit anti-human CD45 antibody (Sigma Aldrich; SAB4502541). After washing with PBS, samples were incubated with fluorophore-conjugated secondary antibodies (Invitrogen) for 2 hours at room temperature. Slides were covered with cover-slips using ProLong™ Diamond Antifade Mountant with 4’,6-diamidino-2-phenylindole (DAPI) (Thermofisher; P36962). Cells in suspension were cytospun onto glass slides using a Shandon Cytospin cytocentrifuge, fixed with methanol, and stained using a Wright-Giemsa Stain Kit (Abeam; ab245888). Histological stains were imaged using an Olympus Upright Microscope with Slide Scanner (Olympus; BX61V5F) and immunofluorescent images were taken with a Nikon Ti Eclipse inverted microscope at the Columbia HICCC Confocal and Specialized Imaging Core.

[00194] eCT bulk transcriptomics: Cardiac microtissucs were snap frozen and processed by Genewiz/Azenta Bio for RNA extraction, sequencing, and analysis. Briefly, RNA was processed using RNA depletion library' preparation, and sequenced using the Illumina HiSeq 2x150 bp sequencing index. Tissues of poor RIN value (<6) and below usable concentration were not included in analysis (n = 1 control; n = 3 photon; n = 3 neutron). Differentially expressed genes were analyzed using DESeq2 66), and processed with g:Profiler, Revigo, or Cytoscape with top differentially expressed genes (padj<0.05; log2FC>l) for gene ontology analysis.

[00195] Single cell transcriptomic analysis: Cell preparation: Cells were isolated from eBM cultures 21 days post-radiation exposure by collecting the suspension fraction in FACS buffer and counted to ensure viability of >90%. Two technical replicates were then mixed at equal amounts at a concentration of 1000 cells per pL. Cells were super-loaded onto the Chromium Next GEM Chip G (PN-1000120) targeting 15,000 cells. Libraries were prepared using the following reagents from the Chromium Single Cell 3’ Reagent Kit (v3.1): Single Cell 3’ Library & Gel Bead Kit v3. 1 (PN-1000121), Chromium Next GEM Chip G Single Cell Kit (PN-1000120) and Single Index Kit T Set A (PN-1000213) (lOx Genomics). The Chromium Next GM Single Cell 3’ Reagent Kits v3.1 User Guide with Feature Barcoding technology for Cell Surface Protein (CG000206 Rev D) was followed for GEM generation, cDNA amplification and library construction. Libraries were run on either an Illumina HiSeq 4000 as 150-bp paired-end reads at a sequencing depth of at least 20,000 read pairs per cell for the 3’ Gene Expression library and 5,000 read pairs per cell for the Cell Surface Protein library.

[00196] Single cell transcriptomic analysis: Preprocessing and clustering. CellRanger was applied on the raw sequencing data to generate unique molecular identifier (UMI) matrix. The UMI matrix was then imported into Scanpy. Uniform Manifold Approximation and Projection (UMAP) for dimensionality reduction was performed with Leiden algorithm-based clustering prior to any cell filtering to first identify granulocytes, which require a separate preprocessing method as they have relatively lower RNA content. For all cell types, cells expressing less than 200 genes and expressed in less than 3 cells were excluded from analysis. Mitochondrial and ribosomal DNA was excluded. Cells with over 20% mitochondrial content, indicated by the fraction of mitochondrial transcripts over the total transcript counts, were removed from further analysis. Cells were filtered out for low library size of 500 for granulocytes and 1000 for all other cell types. 15,000 most highly variable genes were identified for further analysis. Principal component analysis (PCA) was performed with 50 components, and nearest neighbors were identified with 12 nearest neighbors. UMAP was generated and Leiden clustering was applied on the filtered dataset to visualize and identify specific cell types. To annotate each cluster, previous studies were referenced to identify marker genes expressed. Differential gene expression analyses were done using the Python package Scanpy. Wilcoxon's t-test was applied to rank differential genes. P-values and log fold changes were exported and used for gene ontology analyses with either g:Profiler, Revigo, or Cytoscape using top differentially expressed genes (p<0.05; log2FC>l).

[00197] Single cell transcriptomic analysis: CellChat. CellChat was used for inferring cell-cell communications through analyzing the ligand and receptor signaling pairs from the single cell transcriptomics. Based on mass action models, social network analysis, pattern recognition methods and manifold learning, CellChat is able to identify the specific signaling roles and determine the intercellular communication among different cell populations and sample groups. After annotating the cell identities for each group in our datasets, we constructed the CellChat objects for OGy, 4Gy photon, and IGy neutron respectively. And three types of communication including ECM-receptor, secreted signaling, and cell-cell contacts were analyzed separately and compared among different radiation affected groups.

[00198] Statistics: All cardiac imaging functional data, bone marrow flow cytometry, cytokine, and individual cardiac gene expression data were plotted using GraphPad Prism. All statistical analyses for these were performed using GraphPad Prism with One Way or Two Way ANOVA with Multiple Comparisons, as indicated in each Figure. Cardiac Bulk RNA sequencing differential gene expression analysis was analyzed with Wald Chi-Squared tests for significance. Single-cell RNA sequencing analysis from bone marrow cultures were analyzed using Scanpy’s Rank Genes function using t-tests with Bonferroni corrections.

[00199] Multi -tissue study

[00200] Multi -organ engineered platforms as described in WO2021/237195 were used. Briefly, all components of the bioreactors were autoclaved for sterilization, and assembled using sterile gloves in a Biosafety Cabinet. 8 pm PET membrane meshes were sterilized with 70% ethanol for 24 hours, and washed 2X with IX PBS. GFP-labeled human umbilical vein endothelial cells (HUVECs; cAP-0001GFP, Angio- Proteomie) and iMSCs were collected, counted, and resuspended at a 5: 1 ratio prior to seeding. Endothelial meshes were coated with 1 ug/mL Fibronectin (356008, Corning) for 45 minutes and washed with IX PBS prior to cell seeding. 5 x 10 ⁵ cells per barrier were seeded for 1.5 hours prior to rehydration with EGM-2 endothelial medium (Lonza). Barriers were maintained in static culture for 24 hours, and then transferred into the multi-organ platforms. At this point, barriers were exposed to ramping shear stress up to 1 .88 dynes/cm ² over three days. In parallel, eBMs were matured for two days prior to integration with the inclusion of hematopoietic progenitors from cither iPS or CB sources (i.c. iBM or CB-BM). At the point of integration, eBM and eCT tissues were transferred into multi-organ platforms, with their respective mediums for the duration of the study. Integrated platforms were maintained for three days prior to cryoinjury in healthy state.

[00201] At the point of injury, eCTs from one chamber were removed from the integrated platforms, exposed to 5 seconds of dry ice on one side of the tissue, and then returned to the integrated platforms. All integrated platforms were maintained for another 10 days post-injury and analyzed using flow cytometry, histological staining, and cytokine secretion.

[00202] Dissociation and flow cytometry of cells from eCTs, endothelial barriers, and circulation compartments eCTs, endothelial barriers, and eBMs were removed from the integrated platforms for digestion. eCTs were digested with 12 mg/mL collagenase II (Worthington, LS004176) and 12 mg/mL Nattokinase (Japan Bio Science Lab) for 40-45 minutes at 37 °C. eBMss were digested with 10 mg/mL collagenase II (Worthington, LS004176) and 10 mg/mL Nattokinase (Japan Bio Science Lab) for 1 hour at 37 °C. Endothelial barriers were washed IX with PBS, and treated with TrypLE for 10-15 minutes at 37 °C. Circulating compartment cells were washed out of the circulating tubing by FACS buffer administered through a 20 cc syringe. All samples were washed with FACS buffer 1-2X after digestion. Samples were collected in eppendorfs and filtered with Flowmi® Cell Strainers (Millipore Sigma, BAH136800040), and re-suspended in a 96-well V-Bottom plate (Coming, 3894). All flow cytometry protocols remain the same as prior, except the panel was reduced to one myeloid-specific panel: Alexa 700 CD45, BV421 CDl lb, BV605 CD14, PE CD16, BUV395 CDl lc, APC CD15, BV711 CD13, and Propidium Iodide. Samples were analyzed and plotted using FlowJo and GraphPad Prism, separately for each type of sample (i.e. eCTs versus eBMs versus circulating cells).

[00203 J While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Various alternatives to the specific embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents are covered thereby.