scholarly journals Bone marrow-on-a-chip: Emulating the human bone marrow

2018 ◽  
Author(s):  
Stefan Sieber ◽  
Annika Winter ◽  
Johanna Wachsmuth ◽  
Rhiannon David ◽  
Maria Stecklum ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Culture System ◽  
3D Culture ◽  
In Vitro Cultivation ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem

AbstractMultipotent hematopoietic stem and progenitor cells HSPC reside in specialized stem cell niches within the bone marrow, that provide a suitable microenvironment for lifelong maintenance of the stem cells. Meaningful in vitro models recapitulating the in vivo stem cell niche biology can be employed for both basic research as well as for applied sciences and represent a powerful tool to reduce animal tests in preclinical studies. Recently we published the generation of an in vitro bone marrow niche model, capable of long-term cultivation of HSC based on an organ-on-a-chip platform. This study provides a detailed analysis of the 3D culture system including matrix environment analysis by SEM, transcriptome analysis and system intrinsic differentiation induction. Furthermore, the bone marrow on a chip model can serve to multiply and harvest HSPC, since repeated cell removal not compromised the functionality of the culture system. The prolongation of the culture time to 8 weeks demonstrate the capacity to apply the model in repeated drug testing experiments. The quality of the presented system is emphasized by the differentiation capacity of long-term cultivated HSPC in vitro and in vivo. Transplanted human HSPC migrated actively into the bone marrow of irradiated mice and contributed to the long-term reconstitution of the hematopoietic system after four and eight weeks of in vitro cultivation.The introduced system offers a multitude of possible applications to address a broad spectrum of questions regarding HSPC, the corresponding bone marrow niche biology, and pathological aberrations.

scholarly journals Human fetal bone marrow early progenitors for T, B, and myeloid cells are found exclusively in the population expressing high levels of CD34

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 421-432 ◽  
Author(s):  
D DiGiusto ◽  
S Chen ◽  
J Combs ◽  
S Webb ◽  
R Namikawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Cell Activity ◽  
Hematopoietic Stem ◽  
Fetal Bone ◽  
Cd34 Antigen

Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.

Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2527-2533 ◽  
Author(s):  
RE Ploemacher ◽  
JP van der Sluijs ◽  
CA van Beurden ◽  
MR Baert ◽  
PL Chan
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Linear Regression Analysis ◽  
Correlation Study ◽  
Hematopoietic Stem ◽  
Clonal Assay

Abstract We have developed an in vitro clonal assay of murine hematopoietic precursor cells that form spleen colonies (CFU-S day 12) or produce in vitro clonable progenitors in the marrow (MRA cells) of lethally irradiated mice. The assay is essentially a long-term bone marrow culture in microtiter wells containing marrow-derived stromal “feeders” depleted for hematopoietic activity by irradiation. To test the validity of the assay as a quantitative in vitro stem cell assay, a series of unsorted and physically sorted bone marrow cells were simultaneously assayed in vivo and overlaid on the feeders in a range of concentrations, while frequencies of cells forming hematopoietic clones (cobblestone area forming cells, CAFC) were calculated by means of Poisson statistics. Linear regression analysis of the data showed high correlations between the frequency of CFU-S day 12 and CAFC day 10, and between MRA cells and CAFC day 28. A majority of MRA activity and CAFC day 28 was separable from CFU-S day 12 and CAFC day 10. This correlation study validates the CAFC system as a clonal assay facilitation both the quantitative assessment of a series of subsets in the hematopoietic stem cell hierarchy and the study of single long-term repopulating cells in vitro.

scholarly journals Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2527-2533 ◽  
Author(s):  
RE Ploemacher ◽  
JP van der Sluijs ◽  
CA van Beurden ◽  
MR Baert ◽  
PL Chan
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Linear Regression Analysis ◽  
Correlation Study ◽  
Hematopoietic Stem ◽  
Clonal Assay

We have developed an in vitro clonal assay of murine hematopoietic precursor cells that form spleen colonies (CFU-S day 12) or produce in vitro clonable progenitors in the marrow (MRA cells) of lethally irradiated mice. The assay is essentially a long-term bone marrow culture in microtiter wells containing marrow-derived stromal “feeders” depleted for hematopoietic activity by irradiation. To test the validity of the assay as a quantitative in vitro stem cell assay, a series of unsorted and physically sorted bone marrow cells were simultaneously assayed in vivo and overlaid on the feeders in a range of concentrations, while frequencies of cells forming hematopoietic clones (cobblestone area forming cells, CAFC) were calculated by means of Poisson statistics. Linear regression analysis of the data showed high correlations between the frequency of CFU-S day 12 and CAFC day 10, and between MRA cells and CAFC day 28. A majority of MRA activity and CAFC day 28 was separable from CFU-S day 12 and CAFC day 10. This correlation study validates the CAFC system as a clonal assay facilitation both the quantitative assessment of a series of subsets in the hematopoietic stem cell hierarchy and the study of single long-term repopulating cells in vitro.

scholarly journals Human fetal bone marrow early progenitors for T, B, and myeloid cells are found exclusively in the population expressing high levels of CD34

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 421-432 ◽  
Author(s):  
D DiGiusto ◽  
S Chen ◽  
J Combs ◽  
S Webb ◽  
R Namikawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Cell Activity ◽  
Hematopoietic Stem ◽  
Fetal Bone ◽  
Cd34 Antigen

Abstract Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.

scholarly journals SHP-1 regulates hematopoietic stem cell quiescence by coordinating TGF-β signaling

2018 ◽  
Vol 215 (5) ◽  
pp. 1337-1347 ◽  
Author(s):  
Linjia Jiang ◽  
Xue Han ◽  
Jin Wang ◽  
Chen Wang ◽  
Xiaoqiang Sun ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Stem Cell Quiescence ◽  
Β Receptor ◽  
Signaling Activation

Cell cycle quiescence is critical for hematopoietic stem cell (HSC) maintenance. TGF-β signaling in bone marrow niche has been identified in regulating HSC quiescence; however, the intrinsic regulatory mechanisms remain unclear. This study reports that Shp-1 knockout HSCs have attenuated quiescence and impaired long-term self-renewal. SHP-1–activated HSCs are surrounded by megakaryocytes, which regulate HSC quiescence by producing TGF-β1. Mechanistically, SHP-1 interacts with the immunoreceptor tyrosine-based inhibition motif on TGF-β receptor 1 and is critical for TGF-β signaling activation in HSCs. Functionally, Shp-1 knockout HSCs do not respond to TGF-β–enforced HSC quiescence regulation, both in vitro and in vivo. Therefore, we identify TGF-β–SHP-1 as a novel intrinsic regulatory mechanism for HSC quiescence maintenance.

scholarly journals Closer to Nature: The Role of MSCs in Recreating the Microenvironment of the Hematopoietic Stem Cell Niche in vitro

2022 ◽  
pp. 1-10
Author(s):  
Patrick Wuchter ◽  
Anke Diehlmann ◽  
Harald Klüter
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Niche ◽  
Model Systems ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Hematopoietic Stem Cell Niche ◽  
Cell Niche

<b><i>Background:</i></b> The stem cell niche in human bone marrow provides scaffolds, cellular frameworks and essential soluble cues to support the stemness of hematopoietic stem and progenitor cells (HSPCs). To decipher this complex structure and the corresponding cellular interactions, a number of in vitro model systems have been developed. The cellular microenvironment is of key importance, and mesenchymal stromal cells (MSCs) represent one of the major cellular determinants of the niche. Regulation of the self-renewal and differentiation of HSPCs requires not only direct cellular contact and adhesion molecules, but also various cytokines and chemokines. The C-X-C chemokine receptor type 4/stromal cell-derived factor 1 axis plays a pivotal role in stem cell mobilization and homing. As we have learned in recent years, to realistically simulate the physiological in vivo situation, advanced model systems should be based on niche cells arranged in a three-dimensional (3D) structure. By providing a dynamic rather than static setup, microbioreactor systems offer a number of advantages. In addition, the role of low oxygen tension in the niche microenvironment and its impact on hematopoietic stem cells need to be taken into account and are discussed in this review. <b><i>Summary:</i></b> This review focuses on the role of MSCs as a part of the bone marrow niche, the interplay between MSCs and HSPCs and the most important regulatory factors that need to be considered when engineering artificial hematopoietic stem cell niche systems. <b><i>Conclusion:</i></b> Advanced 3D model systems using MSCs as niche cells and applying microbioreactor-based technology are capable of simulating the natural properties of the bone marrow niche more closely than ever before.

Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1293-1293
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1387-1387
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Integrin Alpha 6

Abstract Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

Leukemia Bone Marrow Primary Cells Long-Term Culture in a Biomimetic Hematopoietic Niche.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4114-4114
Author(s):  
Li Hou ◽  
Ting Liu ◽  
Jing Tan ◽  
Wentong Meng ◽  
Li Deng
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Culture System ◽  
3D Culture ◽  
Primary Cells ◽  
Marrow Mesenchymal Stem Cells ◽  
3D Culture System ◽  
2D System

Abstract We have constructed a biomimetic hematopoietic niche (3D culture system) with bio-derived bone as framework, composited with human marrow mesenchymal stem cells, and induced the cells into osteoblasts. Our primary results showed that the biomimetic 3D culture system is capable to allow maintenance and expansion of primitive hematopoietic progenitor cells in vitro. But so far, leukemia primary cells long-term culture from patients marrow are still difficult because it is not clear how does the regulation of leukemic cells grow ex vivo, and lack of adequate investigation between leukemic stem cells with stromal cells. Based on our previous research, we cultured bone marrow mesenchymal stem cells from chronic myelogenous leukemia (CML) patients, and conceived a “pathologic biomimetic osteoblast niche”, to explore the growth of leukemia bone marrow primary cells from CML patients. Bio-derived bone was composited with marrow mesenchymal stem cells from CML patients and constructed a 3D biomimetic osteoblast niche. The mononuclear cells (MNCs) were collected with standard Ficoll-Paque separation from newly diagnosed CML patients. The MNCs were cultured for 2∼5 weeks in the 3D culture system and compared with 2D culture system. The results showed that the proportion of CD34+ cells are increased either in 3D or 2D culture systems. Compared to input, the proportion of CD34+ cells were increased 6.52(1.87∼9)vs. 3.18(1.07∼6.8)times at 2 weeks culture, and 13.6(3.59∼26.31)vs. 7.86(0.78∼18.0)times at 5 weeks culture. The proportion of CD34+/CD38- was higher in 3D culture system than 2D system. It was 5.55(2.1∼11.7)% vs. 2.4(0.9∼3.4)%, and 13.5(3.4∼34.2)% vs. 4.83(2.1∼8.9)% at 2 weeks and 5 weeks respectively. The function of cultured cells was evaluated in colony forming unit (CFU) assay and long term culture initial cell (LTC-IC) assay. 3D system produced more colonies than 2D system {103.33(82∼144)vs. 79(53∼122)} at 2 week culture and 47(33∼66)vs. 21.67(16∼27)at 5 week culture. LTC-IC are widely used as a surrogate in vitro culture for pluripotent stem cells, and those primitive progenitor cells responsible for leukemia in mice are named SL-IC or leukemia stem cells (LSCs). 3D system showed higher frequency of LTC-IC than that of 2D system after 2-week culture(2.23E-05(1.73∼2.56)vs.1.40E-05(1.21∼1.73)). FISH showed the proportion of Ph+ cells declined in both system during the culture, but not as rapidly as it did in 2D system{65%(3D)vs.63%(2D)at 2 week, 55%(3D)vs.35%(2D)at 5 week}, and the Ph+ cells were predominant derived from 3D culture. Our 3D culture system constructed with induced osteoblasts from mesnchymal stem cells in CML patients might provide a more suitable microenvironment for leukemic cells growing in vitro. The leukemic stem cells seemed to be regulated by the molecular signals mediated by osteoblast, and the biological characteristics of leukemia stem cells at least partially is maintained. It may be become a new method for studying leukemic HSCs/HPCs behavior in vitro.

Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2476-2476
Author(s):  
Kasia Mierzejewska ◽  
Ewa Suszynska ◽  
Sylwia Borkowska ◽  
Malwina Suszynska ◽  
Maja Maj ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Sex Hormones ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Clonogenic Potential

Abstract Background Hematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and Methods To address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. Results We found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. Conclusions Our data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories. Disclosures: No relevant conflicts of interest to declare.

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