This research was supported by the Basic Science Center Program (No. T2288102), the Key Program of the National Natural Science Foundation of China (No. 32230059), the National Natural Science Foundation of China (No. 32301123), the Foundation of Frontiers Science Center for Materiobiology and Dynamic Chemistry (No. JKVD1211002), the Wego Project of Chinese Academy of Sciences (No. (2020) 005), the Project of National Facility for Translational Medicine (Shanghai) (No. TMSK-2021-134), and the China Postdoctoral Science Foundation (No. 2022M721147).

Osteo-Organoid-Derived Cells for Effective HSCT in Irradiated Mice

<ol>
	<li>Copelan, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem-cell+transplantation.">Hematopoietic stem-cell transplantation.</a> New England Journal of Medicine. 354 (17), 1813-1826 (2006).</li><li>Vittayawacharin, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+hematopoietic+stem+cell+transplantation+for+a+patient+with+multiple+autoimmune+diseases.">Autologous hematopoietic stem cell transplantation for a patient with multiple autoimmune diseases.</a> American Journal of Hematology. 98 (10), 1659-1662 (2023).</li><li>Xu, L. -. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+transplantation+activity+in+China+2019:+a+report+from+the+Chinese+Blood+and+Marrow+Transplantation+Registry+Group.">Hematopoietic stem cell transplantation activity in China 2019: a report from the Chinese Blood and Marrow Transplantation Registry Group.</a> Bone Marrow Transplantation. 56 (12), 2940-2947 (2021).</li><li>Gratwohl, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+transplantation:+a+global+perspective.">Hematopoietic stem cell transplantation: a global perspective.</a> JAMA. 303 (16), 1617-1624 (2010).</li><li>Sakurai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+cytokine-free+expansion+of+human+haematopoietic+stem+cells.">Chemically defined cytokine-free expansion of human haematopoietic stem cells.</a> Nature. 615 (7950), 127-133 (2023).</li><li>Pineault, N., Abu-Khader, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+umbilical+cord+blood+stem+cell+expansion+and+clinical+translation.">Advances in umbilical cord blood stem cell expansion and clinical translation.</a> Experimental Hematology. 43 (7), 498-513 (2015).</li><li>Liang, G., Liu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+expansion+of+human+hematopoietic+stem+cells.">Long-term expansion of human hematopoietic stem cells.</a> Cell Regeneration. 12 (1), 18 (2023).</li><li>Wilkinson, A. C., Igarashi, K. J., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+cell+self-renewal+in+vivo+and+ex+vivo.">Haematopoietic stem cell self-renewal in vivo and ex vivo.</a> Nature Reviews Genetics. 21 (9), 541-554 (2020).</li><li>Kumar, S., Geiger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSC+niche+biology+and+HSC+expansion+ex+vivo.">HSC niche biology and HSC expansion ex vivo.</a> Trends in Molecular Medicine. 23 (9), 799-819 (2017).</li><li>Dai, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+BMP-2-triggered+in+vivo+osteo-organoid+for+cell+therapy.">A BMP-2-triggered in vivo osteo-organoid for cell therapy.</a> Science Advances. 9 (1), 1541 (2023).</li><li>Dai, K., Zhang, W., Deng, S., Wang, J., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfated+polysaccharide+regulates+the+homing+of+HSPCs+in+a+BMP-2-triggered+in+vivo+osteo-organoid.">Sulfated polysaccharide regulates the homing of HSPCs in a BMP-2-triggered in vivo osteo-organoid.</a> Advanced Science. 10 (24), e2301592 (2023).</li><li>Alexander, T., Greco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+transplantation+and+cellular+therapies+for+autoimmune+diseases:+overview+and+future+considerations+from+the+Autoimmune+Diseases+Working+Party+(ADWP)+of+the+European+Society+for+Blood+and+Marrow+Transplantation+(EBMT).">Hematopoietic stem cell transplantation and cellular therapies for autoimmune diseases: overview and future considerations from the Autoimmune Diseases Working Party (ADWP) of the European Society for Blood and Marrow Transplantation (EBMT).</a> Bone Marrow Transplantation. 57 (7), 1055-1062 (2022).</li><li>Ding, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+haploidentical+HSCs+combined+with+allogenic+MSCs+for+the+treatment+of+ALL+patients.">Infusion of haploidentical HSCs combined with allogenic MSCs for the treatment of ALL patients.</a> Bone Marrow Transplantation. 57 (7), 1086-1094 (2022).</li><li>Sharrack, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+haematopoietic+stem+cell+transplantation+and+other+cellular+therapy+in+multiple+sclerosis+and+immune-mediated+neurological+diseases:+updated+guidelines+and+recommendations+from+the+EBMT+Autoimmune+Diseases+Working+Party+(ADWP)+and+the+Joint+Accreditation+Committee+of+EBMT+and+ISCT+(JACIE).">Autologous haematopoietic stem cell transplantation and other cellular therapy in multiple sclerosis and immune-mediated neurological diseases: updated guidelines and recommendations from the EBMT Autoimmune Diseases Working Party (ADWP) and the Joint Accreditation Committee of EBMT and ISCT (JACIE).</a> Bone Marrow Transplantation. 55 (2), 283-306 (2020).</li><li>Nelson, M. R., Roy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-marrow+mimicking+biomaterial+niches+for+studying+hematopoietic+stem+and+progenitor+cells.">Bone-marrow mimicking biomaterial niches for studying hematopoietic stem and progenitor cells.</a> J Mater Chem B. 4 (20), 3490-3503 (2016).</li></ol>

The authors declare that they have no competing financial interests.

In this protocol, we present an approach to establish in vivo osteo-organoids with bone marrow-like structures by implanting gelatin sponge scaffolds loaded with BMP-2. We demonstrate that these in vivo osteo-organoids can stably produce therapeutic HSPCs over a long period of time (more than 12 weeks). Compared to existing in vitro expansion or in vivo incubation methods that load cells, this protocol can obtain cell cocktails with diverse cell types, including HSPCs and various immune cells, within 3 weeks10. Animal experiments have also shown that transplantation of in vivo osteo-organoid-derived cells can accelerate hematopoietic recovery in irradiated mice. Given that gelatin sponges loaded with BMP-2 can retain their osteogenic activity for over 12 months after lyophilization when stored at -20 &#176;C and that the material does not necessitate the incorporation of any cells, this variety of gelatin sponge infused with BMP-2 has the potential to be fashioned into an off-the-shelf product for immediate utilization.
There are two pivotal stages in the protocol that require special attention. First, during the material preparation process, it is crucial to maintain sterility to avoid bacterial contamination, which could result in bone formation failure and the inability to attain in vivo osteo-organoids. Second, after lyophilization, it is imperative to store the material at a low temperature to preserve the activity of BMP-2 protein. Additionally, the process of obtaining single-cell suspensions for transplantation must be carried out in a gentle and prompt manner to ensure cell viability. If the cell suspension is placed on ice, we recommend completing the transplantation within 4 h.
Although various biocompatible materials, such as hydroxyapatite, decalcified bone matrix, and matrix gel, have been reported in the literature as scaffold materials for loading BMP-2 to establish in vivo organoids15, we chose gelatin sponges as the scaffold material due to their availability and clinical translational needs. In addition, we chose to lyophilize the gelatin sponge after loading it with BMP-2 solution. This approach can increase the convenience of use and control the release ability of BMP-2 to a certain extent, reducing the dosage of BMP-2 used. From this perspective, this will significantly reduce the side effects of current clinical products that mainly consist of BMP-2 solution. In the cell transplantation process, in vivo organoid-derived cells can be transplanted after simple filtration without lysis of red blood cells, further simplifying the experimental process. In addition to routine peripheral blood chimerism analysis, we also systematically evaluated the engraftment levels of in vivo osteo-organoid derived cells in multiple solid organs of recipient mice, including bone marrow, spleen, and thymus. This approach will provide a more comprehensive evaluation of the therapeutic potential of in vivo osteo-organoid-derived cells in terms of hematopoietic function.
Although in vivo organoid derived cells can accelerate hematopoietic recovery in mice with bone marrow injuries, the number of HSPCs obtained is still lower than that obtained from single femurs. How to modify the material system to obtain a higher quantity and stronger functional HSPCs is an area that needs further exploration. We recently found that sulfated polysaccharides (SCS), especially sulfated chitooligosaccharides (SCOS), can regulate the function of HSPCs in in vivo osteo-organoids11. This result also suggests that the complexation of BMP-2 with more functional molecules should be a key research direction in the future.
Animal experiments have shown that functional HSPCs can be obtained through the in vivo osteo-organoid approach. However, in large animals such as miniature pigs or macaques, it is still unclear how high doses of BMP-2 can induce functional HSPCs, which is an important step for clinical translation and requires further research.
In summary, the in vivo osteo-organoid approach proposed in this protocol can efficiently and stably produce therapeutic HSPCs in vivo and provide a stable cell source for hematopoietic stem cell transplantation. It is worth exploring more HSPC enhancement factors to benefit more patients. In the following clinical application, we are investigating the use of immediate family members of patients as biogenerators to establish the in vivo osteo-organoid for therapeutic cell provision. This approach has the potential to minimize the risk of immunological rejection and eliminate potential ethical issues.

Hematopoietic stem cell transplantation stands as the conventional therapy for a variety of hematological malignancies, as well as numerous inherited and autoimmune disorders1,2,3,4. Nevertheless, the restricted quantity and origin of hematopoietic stem/progenitor cells (HSPCs) have emerged as a substantial impediment to the clinical implementation of hematopoietic stem cell transplantation (HSCT)5,6.
Large-scale in vitro cell expansion is a commonly employed method for harvesting therapeutic cells5,7. Various studies have developed conditions that stimulate HSPCs to self-renew in vitro, typically through the use of a combination of self-renewal agonists (such as cytokines and growth factors) and serum albumin, resulting in the ex vivo expansion of HSPCs8. However, it is important to note that current methods are still time-consuming and challenging to maintain the self-renewal capacity of expanded HSPCs9.
In contrast to the aforementioned method, harvesting cells in vivo presents a new and innovative strategy. This approach involves the establishment of an in vivo osteo-organoid that mimics the native bone marrow structure10,11. To achieve this, we form in vivo osteo-organoids using bone morphogenetic protein-2 (BMP-2)-loaded gelatin scaffolds to obtain abundant and high-quality autologous cell cocktails, including HSPCs. By therapeutically applying these osteo-organoids, we have successfully treated irradiation damage and demonstrated that HSPCs derived from the osteo-organoids can rapidly and stably reconstitute the experimentally impaired immune system.

<table><tbody><tr><td>AF700-anti-CD11b (M1/70)</td><td>eBioscience</td><td>56-0112-82</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>AF700-anti-Sca-1 (D7)</td><td>BioLegend</td><td>108141</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>APC-anti-CD3e (145-2C11)</td><td>Tonbo</td><td>20-0031-U100</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>bio-anti-CD34 (RAM34)</td><td>eBioscience</td><td>13-0341-82</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>BV421-anti-CD127 (IL-7R&amp;alpha;)</td><td>BioLegend</td><td>135023</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>BV510-anti-CD48 (HM48-1)</td><td>BioLegend</td><td>103443</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>BV711-anti-CD16/32 (93)</td><td>BioLegend</td><td>101337</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>Capillary tube&amp;nbsp;</td><td>Shanghai Huake Labware Co.</td><td>DC616297403604-100mm/0.5mm</td><td></td></tr><tr><td>Cell strainer</td><td>CORNING</td><td>352340</td><td></td></tr><tr><td>Ethanol</td><td>GENERAL-REAGENT</td><td>01158566</td><td></td></tr><tr><td>Ethylenediamine tetraacetic acid (EDTA) solution</td><td>Servicebio</td><td>G1105</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid dipotassium salt dihydrate (EDTA-K2)</td><td>Solarbio</td><td>E8651</td><td></td></tr><tr><td>FITC-anti-CD45.2 (104)</td><td>BioLegend</td><td>109806</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>Flowjo</td><td>Becton, Dickinson &amp;amp; Company</td><td></td><td>A flow cytometry.</td></tr><tr><td>Gelatin sponge</td><td>Jiangxi Xiangen Co.</td><td></td><td>Use under sterile conditions.</td></tr><tr><td>HBSS without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Gibco</td><td>14170112</td><td>HBSS without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+ &lt;/sup&gt;can prevent cell aggregation.</td></tr><tr><td>Hematology analyzer</td><td>Sysmex</td><td>pocH-100i Diff</td><td></td></tr><tr><td>iodine swabs</td><td>Xiangtan Mulan Biological Technology Co., Ltd.</td><td>01011</td><td>To prevent the infection after operation.</td></tr><tr><td>Isoflurane</td><td>RWD</td><td>R510-22-10&amp;nbsp;</td><td>To avoid adverse effects of anesthesia waste gases on the environment and laboratory personnel, a gas recovery system should be used in conjunction.</td></tr><tr><td>Kraft paper</td><td></td><td></td><td>absorbent paper</td></tr><tr><td>LIVE/DEAD Fixable Near IR Dead Cell Staining Kit(used in 3.4.5)</td><td>Thermo Fisher Scientific</td><td>L34962</td><td>A live/dead staining kit. Store at -20 &amp;deg;C. Dissolve in 50 &amp;mu;L of DMSO for working solution.</td></tr><tr><td>lubricating vet ointment</td><td>Pfizer</td><td></td><td>To prevent dryness and counteract the ocular irritations caused by isoflurane.</td></tr><tr><td>Neutral balsam</td><td>Solarbio</td><td>G8590</td><td></td></tr><tr><td>nylon filter</td><td>Shanghai Shangshai Wire Mesh Manufacturing Co., Ltd.</td><td></td><td>Used for cell filtration.</td></tr><tr><td>Paraffin liquid</td><td>Macklin</td><td>P815706</td><td></td></tr><tr><td>Paraformaldehyde (PFA) solution</td><td>Servicebio</td><td>G1101</td><td>Immersion fixation is used for routine animal tissues. The volume of fixative used is generally 10-20 times the tissue volume, and fixation at room temperature for 24 hours is sufficient.</td></tr><tr><td>PE-anti-CD45.1 (A20)</td><td>BioLegend</td><td>110708</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>PE-CF594-anti-CD135 (A2F10.1)</td><td>BD Biosciences</td><td>562537</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>PE-Cy5-anti-c-kit (2B8)</td><td>BD Biosciences</td><td>105809</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>PE-Cy7-anti-B220 (RA3-6B2)</td><td>BioLegend</td><td>103222</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>PE-Cy7-anti-CD150 (TC15-12F12.2)</td><td>BioLegend</td><td>115914</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>PE-Dazzle594-anti-CD4 (GK1.5)</td><td>BioLegend</td><td>100456</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>Pentobarbital sodium salt</td><td>Sigma-Aldrich</td><td>57-33-0</td><td>Prepare for use at a concentration of 1% (w/v).</td></tr><tr><td>PerCp-Cy5.5-anti-CD8a (53-6.7)</td><td>BioLegend</td><td>100734</td><td>Store at 4 &amp;deg;C. Dilute 1:200 for staining.</td></tr><tr><td>PerCp-Cy5.5-anti-lineage cocktail</td><td>BD Biosciences</td><td>561317</td><td>Store at 4 &amp;deg;C. Dilute 1:10&amp;nbsp; for staining.</td></tr><tr><td>Red blood cell lysis buffer</td><td>Beyotime</td><td>C3702</td><td>Store at 4 &amp;deg;C. Use in clean bench.</td></tr><tr><td>rhBMP-2</td><td>Shanghai Rebone Biomaterials Co.</td><td></td><td>The concentration of rhBMP-2 in the stock solution is 1.0 mg/mL.</td></tr><tr><td>Staining buffer</td><td>BioLegend</td><td>420201</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Xylene</td><td>GENERAL-REAGENT</td><td>01018114</td><td></td></tr><tr><td>Zombie UV Fixable Viability Kit (used in 6.2.5)</td><td>BioLegend</td><td>423108</td><td>A live/dead staining kit. For reconstitution, bring the kit to room temperature; add 100 &amp;micro;L of DMSO to one vial of Zombie UV dye until fully dissolved.&amp;nbsp;</td></tr></tbody></table>

in vivo osteo-organoid approach for harvesting therapeutic hematopoietic stem/progenitor cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

As per the protocol, we have created a bioactive scaffold by dripping BMP-2 into a degradable gelatin sponge under sterile conditions. The scaffold was then implanted into the lower limb muscles of mice to establish in vivo osteo-organoids. After an incubation period of 12 weeks, we conducted macroscopic photography, histological analysis, and flow cytometry analysis on the osteo-organoids (Figure 1A). The gelatin sponge was cut into cubes with dimensions of 5 mm x 5 mm x 5 mm, and after absorbing 30 &#181;L of BMP-2 stock solution (1.0 mg/mL), a bioactive scaffold was obtained. The bioactive scaffold retained its original shape even after freeze-drying (Figure 1B). Scanning electron microscopy (SEM) images depict a porous structure within the bioactive scaffold that promotes cell adhesion and proliferation (Figure 1C). The interconnections among various pores could enhance blood vessel sprouting and invasion, facilitating nutrient transport to support cell growth and aiding in osteo-organoid formation. After 12 weeks of induction in vivo, the implanted osteo-organoids exhibited a deep red color (Figure 1D). H&#38;E-stained image showed that the bioactive scaffold induced the formation of a natural bone marrow-like structure composed of osteocytes and bone marrow cells, further confirming the formation of in vivo osteo-organoids (Figure 1E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66026/66026fig01.jpg" /> 
Figure 1: Formation and characterization of in vivo osteo-organoids. (A) Schematic diagram for harvesting and evaluating in vivo osteo-organoids induced by bioactive scaffolds. Freeze-dried gelatin scaffolds loaded with BMP-2 were surgically implanted into the lower limb muscles of mice. The in vivo osteo-organoids were explanted after 12 weeks of incubation for subsequent analysis. (B) Macroscopic images and (C) scanning electron microscope images of the bioactive scaffold. Figure 1A-C are reproduced from Dai et al.11. (D) Macroscopic image and (E) H&#38;E-stained sections of the osteo-organoid 12 weeks after implantation. Figure 1D,E are reproduced from Dai et al.10. n = 4 biological replicates. Bone marrow and new bone are shown. Scale bars = 200 &#181;m (C, right; E), 500 &#181;m (C, left), 1 cm (B,D). Abbreviations: BMP-2 = bone morphogenetic protein-2; H&#38;E = hematoxylin and eosin; BM = bone marrow; NB = new bone. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66026/66026fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To assess the long-term therapeutic cell-generating potential of osteo-organoids, we analyzed single-cell suspensions from the osteo-organoids using flow cytometry for quantitative analysis of HSPCs (Supplemental Figure S1). We analyzed the types of HSPC subsets 12 weeks after implantation (Figure 2A). Hematopoietic stem cells (HSCs; Lin&#8722;c-kit+Sca-1+CD48&#8722;CD150+) and their differentiated hematopoietic progenitor cells (HPCs), including multipotent progenitors (MPPs; Lin&#8722;c-kit+Sca-1+CD48&#8722;CD150&#8722;), common lymphoid progenitors (CLPs; Lin&#8722;c-kit-Sca-1-Flt3+IL7R&#945;+), common myeloid progenitors (CMPs; Lin&#8722;c-kit+Sca-1&#8722;CD34+FC&#947;R&#8722;), granulocyte-monocyte progenitors (GMPs; Lin&#8722;c-kit+Sca-1&#8722;CD34+FC&#947;R+), and megakaryocyte erythroid progenitors (MEPs; Lin&#8722;c-kit+Sca-1&#8722;CD34&#8722;FC&#947;R&#8722;) were all present in the osteo-organoids. We then conducted further quantitative analysis of the cell numbers in different cell populations within the osteo-organoids (Figure 2B). After 12 weeks of implantation, the numbers of HSCs, MMPs, CLPs, CMPs, GMPs, and MEPs in the osteo-organoids were approximately 530, 860, 2750, 80,000, 43,000, and 55,000, respectively. In conclusion, these results supported that osteo-organoids contained an abundant supply of HSPCs for cell therapy.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66026/66026fig02.jpg" /> 
Figure 2: In vivo osteo-organoids contain abundant HSPCs. (A) Representative flow cytometric plots of HSPCs in the osteo-organoids 12 weeks after implantation. (B) Absolute numbers of total cells, Hematopoietic stem cells (Lin&#8722;c-kit+Sca-1+CD48&#8722;CD150+) and differentiated hematopoietic progenitors: multipotent progenitors (Lin&#8722;c-kit+Sca-1+CD48&#8722;CD150&#8722;), common lymphoid progenitors (Lin&#8722;c-kit-Sca-1-Flt3+IL7R&#945;+), common myeloid progenitors (Lin&#8722;c-kit+Sca-1&#8722;CD34+FC&#947;R&#8722;), granulocyte-monocyte progenitors (Lin&#8722;c-kit+Sca-1&#8722;CD34+FC&#947;R+), and megakaryocyte erythroid progenitors (Lin&#8722;c-kit+Sca-1&#8722;CD34&#8722;FC&#947;R&#8722;). Figure 2A,B was modified from Dai et al.10. n = 6 biological replicates. Data are presented as means &#177; SD. Statistical differences among groups are identified by one-way ANOVA, followed by Tukey&#39;s multiple comparison tests. *P &#60; 0.05, **P &#60; 0.01, and ***P &#60; 0.001. Abbreviations: HSPCs = hematopoietic stem/progenitor cells; HSCs = Hematopoietic stem cells; MPPs = multipotent progenitors; CLPs = common lymphoid progenitors; CMPs = common myeloid progenitors; GMPs = granulocyte-monocyte progenitors; MEPs = megakaryocyte erythroid progenitors; PE = phycoerythrin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66026/66026fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
HSCT has proven to be an effective cell therapy for treating radiation-induced immune system injury12,13,14. We transplanted cells derived from in vivo osteo-organoids (incubated for 12 weeks) into sublethally irradiated mice and collected samples from different parts of the mice at indicated time points for subsequent analysis (Figure 3A). Hematological analysis showed that, compared to the native BM-treated group, the WBC count in the 5 Gy osteo-organoid-treated group significantly increased at the 4th week post transplantation, suggesting an enhanced WBC recovery capability of the osteo-organoid-derived cells (Figure 3B). In the second week after transplantation, the RBC count of both the 5 Gy osteo-organoid-treated group and the 5 Gy native BM-treated group returned to normal levels (Figure 3C). At 2 weeks post transplantation, the PLT count was significantly increased in the 5 Gy osteo-organoid-treated group and the 5 Gy native BM-treated group compared to the 5 Gy PBS-treated group (Figure 3D). In conclusion, these results confirmed that cells derived from osteo-organoids exerted a more robust capacity to enhance hematopoietic recovery compared to native BM cells.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66026/66026fig03.jpg" /> 
Figure 3: Cell therapy from osteo-organoids for the treatment of sublethally irradiated mice. (A) Experimental scheme of HSCT. The osteo-organoids were generated by implanting bioactive scaffolds into the femur muscle pocket of mice (donors, CD45.1) for 12 weeks. The osteo-organoids were collected and digested to obtain a whole bone marrow cell suspension, which was then transplanted to WT mice (recipients, CD45.2) with immune system injury induced by 5 GY X-ray. peripheral blood was collected at 2, 4, 8, 12, and 16 weeks after transplantation for subsequent analysis of peripheral blood chimerism. Solid organ was collected at 16 weeks after transplantation for subsequent analysis of solid organ chimerism. (B-D) peripheral blood cells from the four groups were analyzed at indicated time points. WT mice in the 0 Gy PBS-treated group did not receive irradiation and received PBS. WT mice in the 5 Gy PBS-treated, 5 Gy native BM-treated, and 5 Gy osteo-organoid-treated groups received 5 Gy irradiation 1 day prior to transplantation and were transplanted with PBS, native BM cells, or osteo-organoid-derived cells, respectively. n = 4 to 5 biological replicates. Figure 3A-D was modified from Dai et al.10. Data are presented as means &#177; SD. (B-D) Significant differences among groups are identified by two-way ANOVA followed by Bonferroni&#39;s post hoc test. **P &#60; 0.01. Abbreviations: HSCT = Hematopoietic stem cell transplantation; BMP-2 = bone morphogenetic protein-2; WT = wild type; PB = peripheral blood; PBS = phosphate-buffered saline; BM = bone marrow; WBC = white blood cell; RBC = red blood cell; PLT = platelet. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66026/66026fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To assess the contribution of transplanted cells to the peripheral immune system reconstitution in irradiated mice, we performed quantitative analysis of CD3+ T cells, B220+ B cells, and CD11b+ myeloid cells using flow cytometry. This allowed us to evaluate the chimerism of osteo-organoid-derived and native BM-derived cells in the peripheral blood of the recipients. (Figure 4A and Supplemental Figure S2). We collected peripheral blood samples at 2, 4, 8, 12, and 16 weeks after cell therapy in different groups (Figure 3A). We observed sustained high levels of chimerism in the peripheral blood, with donor-derived CD3+ T cells, B220+ B cells, and CD11b+ myeloid cells consistently exceeding 80% in both the 5 Gy osteo-organoid treated groups and the 5 Gy native BM treated groups. (Figure 4B and Figure 4E,F). Consistent with hematological analysis, the osteo-organoid-derived cells exhibited enhanced regenerative capacity in the T cell subset chimerism in comparison to the native BM-derived cells (Figure 4C,D). The CD3+ T cell subset from the donor source achieved stable chimerism at 8 weeks post transplantation, while the B220+ B cell and CD11b+ myeloid cell subsets from the donor source achieved stable chimerism as early as 2 weeks after transplantation (Figure 4B-F). These data substantiated the role of osteo-organoid-derived cells from the donor in accelerating the recovery of T cell subsets in peripheral blood compared to the native BM-derived cells from the donor.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66026/66026fig04.jpg" /> 
Figure 4: Reconstitution of the peripheral immune system in sublethally irradiated mice by osteo-organoid-derived cells. (A) The flow cytometric gating strategy for the chimerism analysis of recipient mice peripheral blood, BM, spleen, and thymus. (B-F) peripheral blood chimerism from the 5 Gy native BM-treated and 5 Gy osteo-organoid-treated groups was analyzed by the proportion of T cells, B cells, and myeloid cells at the indicated time points. Figure 4A-F was reproduced from Dai et al.10. n = 4 to 5 biological replicates. Data are presented as means &#177; SD. (B-F) Significant differences among groups were identified by two-way ANOVA followed by Bonferroni&#39;s post hoc test. *P &#60; 0.05, **P &#60; 0.01, and ***P &#60; 0.001. Abbreviations: PB = peripheral blood; BM = bone marrow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66026/66026fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To assess the regenerative capacity of transplanted cells in the irradiated mice&#39;s solid immune organs, we utilized the same flow cytometry gating strategy to analyze the chimerism of both native BM-derived and osteo-organoid-derived cells in the recipients&#39; BM, spleen, and thymus (Figure 4A). At 16 weeks after cell therapy, the chimerism of T cells derived from donor osteo-organoid cells in recipients&#39; BM was found to be increased, while the chimerism of B cells and myeloid cells was observed to be reduced, as compared to the chimerism of native BM cells from the donor (Figure 5A). Donor cells from both the 5 Gy osteo-organoid-treated group and the 5 Gy native BM-treated group exhibited pronounced chimerism in the spleen and thymus of the recipients, characterized by high levels of T cells, B cells, and myeloid cells (typically exceeding 80%) (Figure 5B,C). These findings provided evidence that osteo-organoid-derived and native BM-derived cells efficiently reconstituted the damaged immune system in mice exposed to irradiation.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66026/66026fig05.jpg" /> 
Figure 5: Reconstitution of the solid immune organ in sublethally irradiated mice by osteo-organoid-derived cells. (A-C) BM, spleen, and thymus chimerism from the 5 Gy native BM-treated and 5 Gy osteo-organoid-treated groups were analyzed by the chimerism rates of T cells, B cells, and myeloid cells at 16 weeks after transplantation. Figure 5A-C were reproduced from Dai et al.10. n = 4 to 5 biological replicates. Data are presented as means &#177; SD. Statistical differences among groups are identified by two-way ANOVA followed by Bonferroni&#39;s post hoc test. *P &#60; 0.05, **P &#60; 0.01, and ***P &#60; 0.001. Abbreviation: BM = bone marrow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66026/66026fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure S1: Gating strategy used to identify HSPCs in the osteo-organoid. Single-cell suspensions from the osteo-organoid were subjected to flow cytometry for quantitative analysis of HSPCs. This figure was reproduced from Dai et al.10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66026/supplement figure 1.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure S2: Gating strategy used to identify donor derived CD45.1+ cells in PB, BM, spleen, and thymus of CD45.2 recipients. Single-cell suspensions from PB, BM, spleen, and thymus of the CD45.2 recipients were analyzed via flow cytometry for the quantitative analysis of donor chimera. This figure was reproduced from Dai et al.10. Abbreviations: PB = peripheral blood; BM = bone marrow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66026/supplement figure 2.pdf" target="_blank">Please click here to download this File.</a>

Read this Scientific Article Protocol about In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells at JoVE.com

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

in-vivo-osteo-organoid-approach-for-harvesting-therapeutic

Nanyang Technological University

Research

JoVE Journal

Bioengineering

384 Views.  East China University of Science and Technology. We zijn al lang bezig met onderzoek naar biomaterialen en introduceren het baanbrekende concept van materiaalbiologen in ontwikkeling van virussen, actieve botherstelmaterialen en de technologen voor groeifactor, voorbereiding en activering. We ontwikkelden een In Vivo Osteo-Organoïde benadering met behulp van bioactieve materialen om on-demand productie van autoknobbeltjes, weefsels en de cellen voor de behandeling van ziekten te bereiken. De traditionele t...

Video: In vivo osteo-organoïde benadering voor het oogsten van therapeutische hematopoëtische stam-/voorlopercellen

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Developmental Biology

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

3D Co-Culture Assay: Vascularized Osteogenic Niches for Cancer Drug Screening

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

Author Spotlight: Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(Ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate  

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Biology

Optimizing Stromal Cell Isolation from Thymus and Bone Marrow

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Author Spotlight: Advancing Hematopoietic Research Using Stromal Cell Isolation for Single Cell Sequencing

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

Immunology and Infection

Intrafemoral Injection for Bone Marrow Study in Live Mice

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

Medicine

A Human Bone Marrow 3D Model to Investigate the Dynamics and Interactions Between Resident Cells in Physiological or Tumoral Contexts

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex extracellular matrix and various cell types, such as mesenchymal stem cells, osteoblasts, and endothelial cells, is deeply involved in hematopoietic stem cell regulation through direct cell-cell interactions, as well as cytokine production. To closely mimic this in vivo structure and conduct experiments reflecting the responses of the human bone marrow, several 3D models have been created based on biomaterials, relying primarily on primary stromal cells. Here, a protocol is described to obtain a minimal and standardized system that is easy to set up and provides features of bone marrow-like structure, which combines different cell populations including endothelial cells, and reflects the heterogeneity of in vivo bone marrow tissue. This 3D bone marrow-like structure-assembled using calcium phosphate-based particles and human cell lines, representative of the bone marrow microenvironment-allows the monitoring of a wide variety of biological processes by combining or replacing different primary cell populations within the system. The final 3D structures can then either be harvested for image analysis after fixation, paraffin-embedding, and histological/immunohistochemical staining for cell localization within the system, or dissociated to collect each cellular component for molecular or functional characterization.

Here, we describe an easy-to-implement, standardized, microphysiological system that reflects the complexity of the human bone marrow's in vivo structure, providing a pertinent model to finely study a broad range of normal and pathological events.

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex ...

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout life and reconstitute the entire blood system following transplantation. HSCs are used clinically in stem cell transplantation therapies, which represent curative treatment for a range of blood diseases. There is substantial interest in both understanding the mechanisms that regulate HSC activity and hematopoiesis, and developing new HSC-based therapies. However, the stable culture and expansion of HSCs ex vivo has been a major barrier in studying these stem cells in a tractable ex vivo system. We recently developed a polyvinyl alcohol-based culture system that can support the long-term and large-scale expansion of transplantable mouse HSCs and methods to genetically edit them. This protocol describes methods to culture and genetically manipulate mouse HSCs via electroporation and lentiviral transduction. This protocol is expected to be useful to a wide range of experimental hematologists interested in HSC biology and hematopoiesis.

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Presented here is a protocol to initiate, maintain, and analyze mouse hematopoietic stem cell cultures using ex vivo polyvinyl alcohol-based expansion, as well as methods to genetically manipulate them by lentiviral transduction and electroporation.

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout ...

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

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