Disease Modeling in iPSC-Derived Human Bone Marrow Organoids

3. Using a wide-bore transfer pipette, aspirate 2 mL of the ice-cold APEL2 medium and gently pipette up and down until the hydrogel is fully dissociated. Transfer the hydrogel fragments containing organoids into a new 15 mL conical tube.

4. Add ice-cold APEL2 medium to a final volume of 8 mL and centrifuge at 200× g for 10 min at 4 °C. The organoids should collect at the bottom of the tube (Figure 7). Carefully aspirate and discard the supernatant containing gel and medium using a wide-bore transfer pipette without disturbing the organoids.

5. Mix the retained conditioned medium with organoid maturation medium at a 1:1 ratio and gently resuspend the organoids in this medium mixture.

6. Using a wide-bore transfer pipette, gently transfer the organoids into an ultra-low-attachment 96-well plate. Ideally, each well should contain a single organoid.

7. Add the remaining conditioned medium/organoid growth medium mixture to the 96-well plate so that each well contains at least 200 μL of medium. Culture for 72 h to allow final organoid maturation (Figure 8).

Note: After this step, the organoids are ready for engraftment with human-derived HSPCs. For renal capsule implantation, continue culturing the organoids in maturation medium until day 21, with half-medium changes every 48 h using organoid maturation medium (aspirate 100 μL of old medium and add 100 μL of fresh medium). By day 21, the organoids become substantially larger and are easier to manipulate surgically.

B. Isolation and storage of patient-derived CD34+ HSPCs

1. Dilute bone marrow aspirate using ice-cold MACS buffer at a 1:5 ratio in a 50 mL Falcon tube.

Note: Bone marrow aspirate samples should not be stored at 4 °C for longer than 72 h, as prolonged storage may lead to coagulation, reduced hematopoietic cell viability, and lower recovery of CD34+ HSPCs after purification.

2. Isolate mononuclear cells by Ficoll-Paque density gradient centrifugation. Add 20 mL of Ficoll-Paque to a 50 mL Falcon tube. Using a 25 mL serological pipette, slowly layer the diluted bone marrow aspirate onto the Ficoll-Paque along the inner wall of the tube at a flow rate of <1 mL/s.

Note: Dispense the diluted bone marrow aspirate using a pipette controller set to its lowest dispensing speed. Dispensing 20 mL should take at least 20 s to maintain a flow rate of <1 mL/s.

3. Carefully transfer the tubes to a centrifuge and spin at 400× g for 30 min at 18 °C using the lowest acceleration and deceleration settings. Avoid disturbing the tubes before and after centrifugation to prevent mixing of the layers.

4. After centrifugation, the mononuclear cells will form an interphase layer beneath the clear supernatant. Using a 10 mL serological pipette, remove the upper clear layer. Then, use a 1 mL pipette tip to carefully aspirate the mononuclear cell layer and transfer it to a 15 mL conical tube.

Note: It is acceptable to conservatively collect a small amount of the upper clear layer and Ficoll-Paque layer together with the mononuclear cells.

5. Add ice-cold MACS buffer to the 15 mL Falcon tube containing the mononuclear cells to a final volume of 10 mL. Centrifuge at 300× g for 10 min at 4 °C. The mononuclear cells will pellet at the bottom of the tube. Carefully remove the supernatant.

6. Resuspend the mononuclear cell pellet in ice-cold MACS buffer and enrich CD34+ HSPCs using the MACS Human CD34 MicroBead kit according to the manufacturer’s instructions. After enrichment, count the cells and proceed immediately to downstream applications or cryopreserve the purified CD34+ cells in CryoStor CSB.

C. Engraftment of CD34+ HSPCs into mature bone marrow organoids

1. Thaw cryopreserved CD34+ HSPCs and determine cell number and viability by Trypan Blue staining using an automated cell counter. If viability is below 90%, remove dead cells using a MACS Dead Cell Removal kit according to the manufacturer’s instructions.

2. Label the cells with CellTrace Far Red kit for donor-cell tracking.

3. Resuspend the labeled cells in 200 μL of ice-cold StemPro-34 medium and keep the cell suspension on ice.

4. Add 100 μL of ice-cold Matrigel to the cell suspension and mix gently. Keep the suspension cold until use. Adjust the cell density such that each 30 μL aliquot contains the desired number of HSPCs for one organoid. For drug sensitivity screening, use 5 × 10³ HSPCs per organoid.

Note: Scale the volume according to the number of organoids to be engrafted.

5. Remove the culture medium from each mature organoid from section A.

6. Apply 30 μL of the cell suspension directly onto each organoid to immerse the organoid.

7. Incubate for 30 min at 37 °C.

8. Add 150 μL of engraftment-support medium to each well.

9. Culture the engrafted organoids for 24 h before drug treatment for drug sensitivity screening.

D. Organoid implantation under the kidney capsule

Note: The kidney organoid implantation protocol was adapted from a method previously described by Nicholson et al. [9].

1. Select intact day-21 bone marrow organoids with regular spherical morphology for implantation. Keep the organoids in sterile PBS or culture medium on ice until use.

2. Prepare a sterile surgical field and sterile instruments, including fine forceps, curved forceps, spring scissors or iris scissors, a needle holder, a scalpel, sterile gauze, cotton-tipped applicators, sterile PBS or saline, 5–0 absorbable suture for body wall closure, and wound clips or non-absorbable suture for skin closure.

3. Induce anesthesia with isoflurane in an induction chamber at 3%–5%, then maintain anesthesia by nose cone at 1%–3% during surgery. Confirm adequate anesthesia by the absence of response to a firm toe pinch.

4. Apply ophthalmic ointment to both eyes to prevent corneal drying during anesthesia.

5. Place the anesthetized mouse on a sterile drape over a heating pad in the right lateral decubitus position with the left flank facing upward. Maintain body temperature throughout the procedure and recovery.

6. Remove hair from the left flank using clippers or a chemical depilatory. Prepare the surgical site with alternating antiseptic scrubs, for example, povidone–iodine followed by 70% ethanol.

7. Using a scalpel or fine scissors, make a 1–1.5 cm skin incision over the left flank. Then, make a second incision through the underlying muscle layer, parallel to the spine, sufficient to expose the kidney (Figure 9).

8. Gently exteriorize the left kidney. Do not grasp the kidney or renal vessels directly. Use curved forceps to gently elevate the kidney through the flank incision or apply gentle abdominal pressure to aid exposure. Keep the kidney moist with warm sterile PBS or saline throughout the procedure.

Critical: Do not allow the renal capsule to dry.

9. Using fine forceps, gently lift the renal capsule and make a small incision in the capsule over the caudal–lateral surface of the kidney using fine spring scissors or iris scissors. The opening should be only large enough to accommodate the organoid.

Critical: Avoid cutting into the renal parenchyma.

10. Using a blunt probe made from a glass Pasteur pipette with the tip fire-polished over a gas flame, gently create a shallow subcapsular pocket by separating the capsule from the kidney surface (Figure 10).

11. Transfer one day-21 bone marrow organoid to the capsule opening using sterile fine forceps. Using the blunt probe, gently advance the organoid cranially within the subcapsular pocket until it is fully positioned under the capsule and away from the capsule opening.

Critical: Avoid rupturing the capsule during advancement of the organoid.

12. Return the kidney gently to the abdominal cavity and confirm that the implanted organoid remains under the capsule and does not extrude from the capsule opening.

13. Close the body wall incision with 5–0 absorbable suture.

14. Close the skin using wound clips or non-absorbable suture (Figure 11).

Note: If wound clips are used, remove them after approximately 5–14 days according to wound healing and institutional guidance.

15. Allow the mouse to recover on a warming pad or under a heat source until fully ambulatory. Continue postoperative monitoring until normal spontaneous movement resumes.

16. Administer postoperative analgesia according to the approved institutional animal protocol. Monitor the mice daily for signs of pain, wound dehiscence, bleeding, infection, decreased mobility, or poor body condition.

17. At the desired endpoint, evaluate implantation success and hematopoietic output by gross examination, histology, and/or flow cytometric analysis.

E. Characterization of organoids by confocal imaging

Note: The three-dimensional imaging protocol for human bone marrow organoids was adapted from the previously published fructose-glycerol-based organoid clearing and imaging method described by Dekkers et al. [10].

1. Transfer mature organoids, with or without patient-derived HSPC engraftment, into a 15 mL Falcon tube using a sterile wide-bore transfer pipette.

Note: Do not combine organoids from different experimental groups in the same tube.

2. Add PBS to a final volume of 5 mL and centrifuge at 200× g for 5 min. Carefully remove the supernatant using a 1 mL pipette tip.

3. Add freshly prepared 4% PFA and fix the organoids at room temperature for at least 2 h.

4. Remove the fixative and add 5 mL of 0.25% Triton X-100 in PBS. Incubate for 30 min at room temperature to permeabilize the organoids.

5. Remove the permeabilization buffer and add blocking buffer (5% goat serum and 0.125% Triton X-100 in PBS). Incubate for 30 min at room temperature.

6. Dilute the primary antibodies and biotinylated UEA1 in blocking buffer at the desired concentrations. Add 250 μL of the antibody solution to one well of a 24-well plate.

Note: Use antibody panels appropriate for the intended analysis. Example antibody panels are provided in Supplemental Table 1 of [5].

7. Transfer the organoid into the well containing the primary antibody solution. Seal the plate with parafilm and incubate at 4 °C on an orbital shaker at 90 rpm for 12 h.

8. Remove the primary antibody solution and wash the organoid with 0.5 mL of 0.125% Triton X-100 in PBS for 15 min. Repeat this wash step three times.

9. Dilute the fluorophore-conjugated secondary antibodies, streptavidin, and DAPI in blocking buffer at the desired concentrations. Add 250 μL of the staining solution to a new well of a 24-well plate.

10. Transfer the organoid into the well containing the secondary antibody solution. Seal the plate with Parafilm and incubate at 4 °C on an orbital shaker at 90 rpm for 12 h.

11. Wash the organoid as described in step E8.

12. Add 0.5 mL of tissue clearing buffer to a new well of a 12-well plate. Using fine forceps, gently hold the organoid at its edge and transfer it into the clearing buffer. Incubate at room temperature for 15 min.

Note: Hold the organoid only at the periphery with the forceps to avoid compressing and distorting its structure. The organoid should become nearly transparent in the clearing buffer. If needed, use a glass Pasteur pipette with a fire-polished blunt tip to gently stir the clearing buffer and locate the organoid.

13. Using a 200 μL pipette tip with the opening cut to approximately 3 mm in diameter, transfer the organoid together with approximately 30 μL of clearing buffer onto an organoid imaging slide.

Note: Detailed methods for preparation of organoid imaging slides can be found in the protocol published by Dekkers et al. [10].

14. Carefully place a coverslip over the organoid and secure the coverslip with tape on both sides.

15. Image the organoids immediately by confocal microscopy.

Note: Representative validation images are provided in Figures 1 and 3 of [5] and in Figure 4K–M of [11].

F. Characterization of organoids with flow cytometry

1. Transfer mature organoids, with or without patient-derived HSPC engraftment, into a 15 mL Falcon tube using a sterile wide-bore transfer pipette.

Note: Do not combine organoids from different experimental groups in the same tube.

2. Add PBS to a final volume of 5 mL and centrifuge at 200× g for 5 min. Carefully remove the supernatant using a 1 mL pipette tip.

3. Add 1 mL of prewarmed 0.25% collagenase to the organoid pellet.

4. Incubate the tube at 37 °C for 15 min. During incubation, gently pipette the suspension up and down every 5 min using a 1 mL pipette tip to facilitate dissociation.

5. After dissociation, pass the cell suspension through a 40-μm cell strainer into a new tube to remove undigested debris.

6. Add MACS buffer to the filtered suspension to a final volume of 5 mL and centrifuge at 300× g for 5 min at 4 °C.

7. Carefully remove the supernatant and resuspend the cell pellet in 200 μL of MACS buffer.

8. Add fluorophore-conjugated primary antibodies at the desired dilutions and incubate the cells for 15 min at room temperature, protected from light.

Note: Use antibody panels appropriate for the intended analysis. Example antibody panels used for flow cytometry characterization are provided in Supplemental Table 2 of [5].

9. Wash the stained cells by adding 1 mL of MACS buffer and centrifuge at 300× g for 5 min at 4 °C.

10. Remove the supernatant and resuspend the pellet in an appropriate volume of MACS buffer for flow cytometry acquisition.

11. Add DAPI immediately before acquisition to exclude dead cells.

12. Acquire the samples on a flow cytometer and analyze the data using FlowJo or equivalent software.

13. Gate singlets and exclude debris and dead cells before downstream analysis of stromal, endothelial, hematopoietic, or donor-derived populations.

Note: For engrafted organoids, first gate on CellTrace+ donor-derived cells, then analyze lineage markers or viability within that population.

14. Quantify the frequency of the desired cell populations and compare across experimental groups.

Note: Representative flow cytometry analyses are provided in Supplemental Figure 3 of [5].

G. Drug sensitivity testing of JAK2V617F-mutant myeloproliferative neoplasm (MPN)-derived HSPCs in engrafted bone marrow organoids

1. For drug sensitivity testing, isolate CD34+ HSPCs from the bone marrow of a patient with JAK2V617F-positive myeloproliferative neoplasm (MPN) and engraft them into mature bone marrow organoids as described in section C. In the published application [8], 5 × 10³ CellVue-labeled donor CD34+ cells were used per organoid.

2. Culture the engrafted organoids for 1 day under normoxic conditions to allow donor-cell lodging within the organoid niche.

3. After engraftment is established, treat the organoids with cyclosporin A (CsA) at 5 or 10 μM, or with DMSO vehicle control, in fresh engraftment-support medium for 3 days.

Note: Users may substitute other compounds or drugs at physiologically relevant concentrations and treatment durations according to the specific goals of their experimental design.

4. At the end of treatment, collect the organoids using a sterile wide-bore transfer pipette and transfer them into a 15 mL Falcon tube.

Note: In the published study, 20 organoids were collected for each flow cytometry assay to improve the robustness of the drug sensitivity analysis. The number of organoids may be adjusted according to experimental design; however, pooling at least eight organoids per flow cytometry assay is recommended.

5. Wash the organoids with PBS and centrifuge at 100× g for 5 min. Carefully discard the supernatant.

6. Add 1 mL of prewarmed collagenase to the organoid pellet and incubate at 37 °C for 15 min. During incubation, gently pipette the suspension up and down every 5 min with a 1 mL pipette tip to facilitate dissociation into single cells. Pass the resulting suspension through a 40-μm cell strainer into a new tube to remove undigested debris.

7. Wash the dissociated cells with PBS or MACS buffer and stain them with fluorophore-conjugated antibodies for flow cytometric analysis of erythroid and myeloid differentiation. In the published MPN application, CellVue was used to identify donor-derived cells, and lineage analysis was performed separately on CellVue-positive donor-derived and CellVue-negative organoid-derived populations.

8. Analyze the samples by flow cytometry. First, gate on CellVue+ donor-derived cells, then quantify erythroid and myeloid populations within the donor-derived compartment using the appropriate lineage markers. In parallel, analyze the CellVue organoid-derived cells as the recipient compartment.

Note: Representative flow analyses can be found in Figure 7E of [8].

9. Compare the lineage output of donor-derived cells between DMSO-treated and CsA-treated groups.

Note: In the published study, CsA treatment caused a dose-dependent reduction in donor-derived erythroid and myeloid output from JAK2V617F-positive patient cells in the organoid model.

H. Using CRISPR/Cas9-edited human CD34+ HSPCs to evaluate the role of DDX41 in erythropoiesis in the bone marrow organoid system

1. Thaw human CD34+ HSPCs and culture 5 × 10⁵ cells in 1 mL of human CD34+ expansion medium (StemCell Technologies, StemSpan SFEM supplemented with StemSpan CD34+ Expansion Supplement).

2. Add approximately 3 × 10⁷ lentiviral particles carrying either DDX41 sgRNA or scrambled sgRNA to 5 × 10⁵ CD34+ cells.

Note: DDX41-knockout HSPCs are used here as an example application of the organoid engraftment assay. Users may manipulate genes of interest in HSPCs using their preferred gene delivery or editing system, such as retroviral or adenoviral vectors or chemical transfection. A detailed method for lentiviral particle production using the lentiCRISPR v2 vector (Addgene #52961) is described in [7].

3. Incubate the cells with lentivirus for 14 h.

4. Wash the infected cells with PBS to remove residual viral particles.

5. Resuspend the cells in fresh human CD34+ expansion medium and allow them to recover for 6 h before organoid engraftment.

6. Label the control and DDX41 sgRNA-transduced CD34+ cells with CellVue according to the manufacturer’s instructions before organoid engraftment. Donor-derived cells were identified as CellVue+ cells after engraftment.

7. Adjust the cell density such that each organoid receives 1 × 10⁴ CellVue-labeled donor CD34+ cells. In the published study [7], 10,000 donor CD34+ HSPCs were co-incubated with each organoid in a 96-well plate.

8. Engraft the donor cells into mature bone marrow organoids as described in section C.

9. Culture the engrafted organoids for three days under normoxic conditions.

10. At the endpoint, collect the organoids using a sterile wide-bore transfer pipette and transfer them into a 15 mL Falcon tube.

11. Pool 10 organoids for each flow cytometry assay. In the published DDX41 organoid experiment, each data point represented cells combined from 10 organoids.

12. Wash the organoids with PBS and centrifuge at 100× g for 5 min. Carefully discard the supernatant.

13. Add 1 mL of prewarmed collagenase to the organoid pellet and incubate at 37 °C for 15 min. During incubation, gently pipette the suspension up and down every 5 min with a 1 mL pipette tip to facilitate dissociation into single cells.

14. Pass the dissociated suspension through a 40-μm cell strainer into a new tube to remove undigested debris.

15. Wash the filtered cells with PBS or MACS buffer and stain them with fluorophore-conjugated antibodies for flow cytometric analysis of donor-derived erythroid and myeloid differentiation.

16. Analyze the samples by flow cytometry. First, gate on CellVue+ donor-derived cells, then quantify lineage output using the appropriate markers. In the published study, CD71 was used to assess erythroid output and CD11b was used to assess myeloid output in donor-derived cells.

17. In parallel, analyze the CellVue-negative organoid-derived cells as the recipient compartment. In the published study, erythroid output from the recipient compartment was also reduced after engraftment of DDX41-deficient donor cells.

18. Compare the frequency of donor-derived CD71+ erythroid cells and CD11b+ myeloid cells between the scrambled sgRNA and DDX41 sgRNA groups. In the published study, DDX41 depletion selectively reduced donor-derived CD71+ erythroid cells, whereas CD11b+ myeloid cells were not significantly reduced.

Note: Representative flow analyses can be found in Figure 7D, F of [7].

Validation of protocol

This protocol has been used and validated in the following research articles:

1. Ren et al. [5]. Development of iPSC-derived human bone marrow organoid for autonomous hematopoiesis and patient-derived HSPC engraftment. Blood Advances. (Figures 1–5)

2. Bi et al. [7]. DDX41 resolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death. Nature Communications. (Figure 7A–G)

3. Wang et al. [8]. PPIL2 is a target of the JAK2/STAT5 pathway and promotes myeloproliferation via degradation of p53. Journal of Clinical Investigation. (Figure 7A–G)

4. Han et al. [11]. Spatial transcriptomic analyses highlight distinct erythroid niches in mice and humans. Nature Genetics. (Figure 4K–M; Figure 6B, C, F)

General notes and troubleshooting

General notes

The general protocol for generating human iPSC-derived bone marrow organoids was adapted from [4]. In our modified protocol, mesoderm/angiogenic induction was performed at 1% O₂ rather than 5% O₂ to provide a measurably stronger hypoxic stimulus and enhance hypoxia-inducible factor-dependent signaling [12], which is important for the subsequent emergence of hemogenic endothelium and hematopoietic stem cell formation [13]. We also included IL-21 during 3D hydrogel culture to support vascular [14] and HSPC expansion [15,16]. Under our conditions, these modifications supported reproducible embryoid body growth and downstream organoid differentiation. Embryoid bodies typically reached approximately 100 μm after 24 h and approximately 200 μm after 72 h of hypoxic induction, and day-21 organoids were suitable for downstream engraftment, imaging, flow cytometry, and renal capsule implantation. Successful mature organoids were typically dense, spherical, and structurally intact.

To facilitate engraftment, donor-derived CD34+ cells were suspended in a small volume of ice-cold Matrigel before seeding onto mature organoids. This step was intended to improve local retention of the cells on the organoid surface and provide a transient basement membrane-like extracellular matrix environment to support early cell attachment and lodging [17]. Downstream engraftment efficiency depends strongly on the quality and viability of patient-derived CD34+ cells. Bone marrow aspirate samples should therefore be processed promptly, and cryopreserved CD34+ cells should be assessed carefully after thawing. For drug sensitivity testing, 5 × 10³ HSPCs per organoid were sufficient for analysis, but at least nine organoids were required for each treatment condition to generate three biological replicates. Because organoid generation and maturation require several weeks, drug screening cannot be performed immediately after iPSC differentiation is initiated. In addition to cell quality, inter-patient differences in HSPC fitness and disease biology may influence the magnitude of ex vivo drug response. Hydrogel preparation is another major technical variable, as incorrect pH adjustment or poor handling of matrix components can compromise organoid formation. Extended drug treatments may require medium replacement and re-addition of compounds depending on drug stability and experimental design.

Another limitation of the current model is that it is not optimized for mature lymphoid output. In particular, full T-cell maturation is not expected because thymic selection requires a specialized thymic microenvironment that is not reproduced in a bone marrow organoid. Although early B-lineage development may be more compatible with a bone marrow-like niche, robust maturation of functional B cells has not been established in the current system. In addition, the current organoid model does not include a mineralized skeletal compartment or mature osteoblasts and osteoclasts. Therefore, while the model recapitulates key endothelial, stromal, and hematopoietic components of the human bone marrow niche, it is not designed to reproduce interactions that specifically depend on mineralized bone or bone-remodeling cells.

Troubleshooting

Problem 1: Embryoid bodies fail to reach the expected size during mesoderm/angiogenic induction.

Possible causes: Poor starting iPSC quality, incorrect cytokine preparation, or unstable hypoxic culture conditions.

Potential solution: Confirm the quality of the starting iPSC culture, prepare fresh induction medium, and verify that the hypoxia incubator is stable at 1% O₂. Under our conditions, embryoid bodies should reach approximately 100 μm after 24 h and approximately 200 μm after 72 h of hypoxic induction.

Problem 2: Patient-derived CD34+ cells show low engraftment efficiency.

Possible causes: Low cell viability after thawing, delayed processing of bone marrow aspirates, insufficient retention of donor cells on the organoid surface, or poor HSPC fitness.

Potential solution: Process bone marrow aspirates promptly and assess the viability of cryopreserved CD34+ cells immediately after thawing. If viability is below 90%, remove dead cells before engraftment. Keep the donor-cell suspension cold after addition of Matrigel and seed the suspension directly onto the organoid surface without delay.

Problem 3: Engrafted donor cells are difficult to detect by flow cytometry.

Possible causes: Low donor-cell input, weak CellVue labeling, poor engraftment, or excessive cell loss during organoid dissociation.

Potential solution: Confirm adequate CellVue labeling before engraftment, use a consistent donor-cell input, and avoid over-digestion or harsh pipetting during collagenase treatment. If the tested drug causes substantial cell death, increase the number of donor cells used for engraftment to ensure sufficient recovery of donor-derived cells for analysis.

Problem 4: Organoids do not dissociate efficiently into single cells for flow cytometry.

Possible cause: Incomplete collagenase digestion or inadequate mechanical disruption.

Potential solution: Use prewarmed collagenase and incubate at 37 °C for the full digestion period. Gently pipette the suspension every 5–10 min during digestion to disrupt the organoid structure. Filter the suspension through a 40-μm strainer before staining.

Problem 5: No human blood cells are detected in NSG mice after renal capsule implantation.

Possible causes: The capsular opening is too large, the capsule is torn during manipulation, or the organoid extrudes when the kidney is returned to the abdominal cavity.

Potential solution: Make the capsular opening only large enough to accommodate the organoid, create a shallow subcapsular pocket before implantation, and gently advance the organoid away from the capsule opening. Confirm that the organoid remains under the capsule before returning the kidney to the abdominal cavity.

Problem 6: Organoids lose their spherical morphology during immunostaining or clearing.

Possible causes: Mechanical compression during transfer or mounting, excessive force with forceps, or distortion under the coverslip.

Potential solution: Handle organoids gently and hold them only at the edge when using forceps. Use wide-bore pipette tips for transfer steps. Avoid grasping the center of the organoid. During mounting, place the coverslip carefully to avoid compressing the structure.

Acknowledgments

This work was supported by the National Cancer Institute (NCI) grant R00CA289959 (K.R.) and EvansMDS Young Investigator Award (K.R.). K.R. is a recipient of the Leukemia & Lymphoma Society (Blood Cancer United) Special Fellow Award.

Author contributions

Conceptualization, E. L., K. R.; Investigation, E. L., A. J.; Writing—Original Draft, A. J., K. R.; Writing—Review & Editing, A. J., A. Z.; Funding acquisition, K. R.; Supervision, K. R., E. L.

Competing interests

K.R. and E.L. are co-founders of MarrowGen and co-inventors on patent application US20260071185A1 related to bone marrow organoid technology. The remaining authors declare no competing interests.

Ethical considerations

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Stony Brook University under protocol IACUC2026-00002 and were performed in accordance with the approved institutional guidelines and regulations.

References

1. Cain, T. L., Derecka, M. and McKinney-Freeman, S. (2024). Author Correction: The role of the haematopoietic stem cell niche in development and ageing. Nat Rev Mol Cell Biol. 26(1): 80–80. https://doi.org/10.1038/s41580-024-00787-z
2. Pinho, S. and Frenette, P. S. (2019). Haematopoietic stem cell activity and interactions with the niche. Nat Rev Mol Cell Biol. 20(5): 303–320. https://doi.org/10.1038/s41580-019-0103-9
3. Frenz-Wiessner, S., Fairley, S. D., Buser, M., Goek, I., Salewskij, K., Jonsson, G., Illig, D., zu Putlitz, B., Petersheim, D., Li, Y., et al. (2024). Generation of complex bone marrow organoids from human induced pluripotent stem cells. Nat Methods. 21(5): 868–881. https://doi.org/10.1038/s41592-024-02172-2
4. Khan, A. O., Rodriguez-Romera, A., Reyat, J. S., Olijnik, A. A., Colombo, M., Wang, G., Wen, W. X., Sousos, N., Murphy, L. C., Grygielska, B., et al. (2022). Human Bone Marrow Organoids for Disease Modeling, Discovery, and Validation of Therapeutic Targets in Hematologic Malignancies. Cancer Discov. 13(2): 364–385. https://doi.org/10.1158/2159-8290.cd-22-0199
5. Ren, K., Li, E., Aydemir, I., Liu, Y., Han, X., Bi, H., Wang, P., Tao, K., Ji, A., Chen, Y. H., et al. (2024). Development of iPSC-derived human bone marrow organoid for autonomous hematopoiesis and patient-derived HSPC engraftment. Blood Adv. 9(1): 54–65. https://doi.org/10.1182/bloodadvances.2024013361
6. Bourgine, P. E. (2026). Human bone marrow organoids: emerging progress but persisting challenges. Trends Biotechnol. 44(1): 11–21. https://doi.org/10.1016/j.tibtech.2025.05.028
7. Bi, H., Ren, K., Wang, P., Li, E., Han, X., Wang, W., Yang, J., Aydemir, I., Tao, K., Ma, R., et al. (2025). DDX41 resolves G-quadruplexes to maintain erythroid genome integrity and prevent cGAS-mediated cell death. Nat Commun. 16(1): e1038/s41467–025–62307–7. https://doi.org/10.1038/s41467-025-62307-7
8. Wang, P., Han, X., Ren, K., Li, E., Bi, H., Aydemir, I., Sukhanova, M., Liu, Y., Yang, J., Ji, P., et al. (2025). PPIL2 is a target of the JAK2/STAT5 pathway and promotes myeloproliferation via degradation of p53. J Clin Invest. 135(13): e1172/jci181394. https://doi.org/10.1172/jci181394
9. Nicholson, T. M., Uchtmann, K. S., Valdez, C. D., Theberge, A. B., Miralem, T. and Ricke, W. A. (2013). Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia. J Visualized Exp. e3791/50574. https://doi.org/10.3791/50574
10. Dekkers, J. F., Alieva, M., Wellens, L. M., Ariese, H. C. R., Jamieson, P. R., Vonk, A. M., Amatngalim, G. D., Hu, H., Oost, K. C., Snippert, H. J. G., et al. (2019). High-resolution 3D imaging of fixed and cleared organoids. Nat Protoc. 14(6): 1756–1771. https://doi.org/10.1038/s41596-019-0160-8
11. Han, X., Ren, K., Wang, P., Bi, H., Li, E., Aydemir, I., Ji, A., Cai, W., Soleimanisardoo, L., Wai, C. M., et al. (2026). Spatial transcriptomic analyses highlight distinct erythroid niches in mice and humans. Nat Genet. 58(7): 1620–1631. https://doi.org/10.1038/s41588-026-02671-2
12. Abaci, H. E., Truitt, R., Luong, E., Drazer, G. and Gerecht, S. (2010). Adaptation to oxygen deprivation in cultures of human pluripotent stem cells, endothelial progenitor cells, and umbilical vein endothelial cells. Am J Physiol, Cell Physiol. 298(6): C1527–C1537. https://doi.org/10.1152/ajpcell.00484.2009
13. Gerri, C., Marass, M., Rossi, A. and Stainier, D. Y. R. (2018). Hif-1α and Hif-2α regulate hemogenic endothelium and hematopoietic stem cell formation in zebrafish. Blood. 131(9): 963–973. https://doi.org/10.1182/blood-2017-07-797795
14. Wang, T., Yang, L., Yuan, M., Farber, C. R., Spolski, R., Leonard, W. J., Ganta, V. C. and Annex, B. H. (2021). MicroRNA-30b Is Both Necessary and Sufficient for Interleukin-21 Receptor-Mediated Angiogenesis in Experimental Peripheral Arterial Disease. Int J Mol Sci. 23(1): 271. https://doi.org/10.3390/ijms23010271
15. Ozaki, K., Hishiya, A., Hatanaka, K., Nakajima, H., Wang, G., Hwu, P., Kitamura, T., Ozawa, K., Leonard, W., Nosaka, T., et al. (2006). Overexpression of Interleukin 21 Induces Expansion of Hematopoietic Progenitor Cells. Int J Hematol. 84(3): 224–230. https://doi.org/10.1532/ijh97.06036
16. Tormo, A., Khodayarian, F., Cui, Y., Al-Chami, E., Kanjarawi, R., Noé, B., Wang, H. and Rafei, M. (2017). Interleukin-21 promotes thymopoiesis recovery following hematopoietic stem cell transplantation. J HematolOncol. 10(1): e1186/s13045–017–0490–3. https://doi.org/10.1186/s13045-017-0490-3
17. Lee-Thedieck, C., Schertl, P. and Klein, G. (2022). The extracellular matrix of hematopoietic stem cell niches. Adv Drug Delivery Rev. 181: 114069. https://doi.org/10.1016/j.addr.2021.114069