Megakaryocytes Exchange Significant Levels of Their Alpha-Granular PF4 with Their Environment

Abstract Platelet factor 4 (PF4, CXCL4) is a major chemokine in megakaryocytes (megs). It is synthesized almost exclusively by megs during their development and may have important roles in regulating both hematopoietic stem cell and megakaryocyte proliferation. We now show that megs both release significant amounts of PF4 into their environment as well as take up PF4 into alpha granules. This PF4 is then available for release by thrombin activation. We examined PF4 recycling during megakaryopoiesis based on the observation that in vitro-cultured human meg hematopoietic precursors release significant amounts of PF4 into the media beginning after approximately 7 days of culture, when definitive megs begin to emerge. Using immunohistochemistry, we find that in vivo in murine bone marrow, human PF4 (hPF4) is released by hPF4 transgenic (hPF4+) megs during the steady-state, and this release is markedly accentuated 48 hours after sub-lethal 660 cGy whole body irradiation from an X-ray source to induce bone marrow injury. By comparison, animals without endogenous PF4 expression (Pf4-/-) showed only background staining. After irradiation, the levels of PF4 staining within the hPF4+ megs decreased with a concomitant increase in background staining suggesting that the stored PF4 was released into the bone marrow milieu. The increase in the PF4 staining in the intramedullary space was not due to released PF4 from entrapped platelets as similar changes were seen in untreated hPF4+ mice and in mice made thrombocytopenic by injection of antiCD41 antibody. We then asked whether the released PF4 could be taken back up by the megs and whether internalized PF4 could reach significant levels compared to endogenously synthesized PF4. We show that murine megs can take up significant levels of hPF4 so that peak hPF4 uptake at 24 hours (19±2 ng/10e6 cells) is equivalent to the amount of mouse (m) PF4 (30±1 ng/106 cells) natively present within the megs. Blocking antibodies to either PF4 itself or to lipoprotein receptor related protein 1 (LRP1) prevented PF4 uptake (53±17 IU/10e6 cells and 32±9 IU/10e6, respectively, vs 95±9 IU/10e6 cells, p <0.01, for either vs. no treatment), consistent with our previous report that LRP1 was necessary for PF4’s negative paracrine effect on megakaryopoiesis. The PF4 that was taken up by megs localizes at least in part to alpha granules, as evidenced by co-localization with P-selectin by immunofluorescence microscopy. Quantification showed a higher degree of colocalization between endogenous mPF4 and internalized hPF4 than between other alpha-granule markers, including vWF, P-selectin and internalized fibrinogen. Moreover like endogenous mPF4, the internalized PF4 can be re-released upon thrombin-induced meg activation. Finally, we asked whether the PF4 uptake was unusual and began by studying uptake of the related chemokine, platelet basic protein (PBP, CXCL7), another protein synthesized by megs and stored in alpha-granules. Unlike PF4, PBP was not internalized by megs as judged by immunohistochemistry or ELISA, indicating that the ability to be internalized and re-released is a relatively unique property of PF4. In summary, we demonstrate that PF4 - an important regulator of megakaryopoiesis and hematopoiesis - is released by megs in the intramedullary space at steady-state and even more so when stressed. Moreover, the released PF4 can be taken up into alpha-granules and stored for potential rerelease. Whether this complex cycle of PF4 in megs is unique to PF4 or applies to other alpha-granular proteins and whether it is necessary for the PF4 effect on hematopoiesis/ megakaryopoiesis needs further investigation Disclosures Xiao: ECRI Institute: Employment.

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High Level Expression of a Membrane-Anchored Inhibitor of HIV Infection in Murine Hematopoietic Stem and Progenitor Cells Does Not Pertubate Serial Multilineage Repopulation.

Blood ◽

10.1182/blood.v104.11.1758.1758 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1758-1758

Author(s):

Axel Schambach ◽

Bernhard Schiedlmeier ◽

Jens Bohne ◽

Dorothee von Laer ◽

Geoff Margison ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Transgene Expression ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Hiv 1 ◽

Marrow Cells

Abstract T20 is a 36-amino-acid peptide that binds to HIV-1 gp41 and thereby acts as a fusion inhibitor, thus mediating potent and selective inhibition of HIV-1 entry in vitro and in vivo. An extended peptide expressed as an artificial, membrane-bound molecule (mbC46) efficiently inhibits HIV infection of primary human T-cells following retroviral vector mediated gene transfer (Egelhofer et al., J Virol, 2004). To develop an even more stringent approach to HIV gene therapy, we targeted hematopoietic stem cells. In 3 experimental groups of C57BL/6 mice (9 animals/group), we investigated the long-term toxicity of murine bone marrow cells transduced with M87o, a therapeutic vector designed to coexpress mbC46 and an HIV-derived RNA RRE-decoy to inhibit HIV replication. As controls we used the same vector containing an inactive C46 peptide and mock-transduced cells. Blood samples were collected monthly. Donor chimerism and transgene expression in multiple lineages were determined by FACS analysis and transgene integration was measured by real time PCR. Six months after transplantation, 4 mice per group were sacrificed and the remaining 5 mice per group were observed for another 6 months. In addition to the parameters mentioned above, we performed complete histopathology, blood counts and clinical biochemistry. Donor chimerism in all groups ranged from 82 – 94% (day 190 and day 349). In the M87o group, 60% of donor cells expressed mbC46. FACS data showed persisting transgene expression in T-cells (CD4, CD8, 65%), B-cells (B220, 46%), myeloid cells (CD11b, 68%), platelets (CD41, 19%), and RBC (60%) of the peripheral blood and bone marrow cells. Highly sustained gene marking (2–4 copies/genome) was noticed on day 190. To reveal latent malignant clones potentially originating from side effects of the genetic manipulation, 1x106 bone marrow cells from 4 primary recipients were transplanted into lethally irradiated secondary recipients (3 recipients/primary mouse) and these mice were observed for 8 months. All together, we could not observe any evidence for leukemogenic capacity. Analysis of peripheral blood and bone marrow showed a similar transgene expression pattern compared to the primary mice. To generate a complete chimerism of transgenic cells, we chose the human drug resistance gene methylguanine-methyltransferase (MGMT, P140K) to select for mbC46-transduced stem cells in vitro and in vivo. Different coexpression strategies were tested. Function of the MGMT protein was confirmed in a quantitative alkyltransferase assay and in a cytotoxicity assay using BCNU or temozolomide. In vitro selection of transduced 32D and PM1 cells with benzylguanine and BCNU showed >95% positive cells with evidence of polyclonal survival. Transduced PM1 cells underwent an HIV challenge assay. In vivo experiments in a murine bone marrow transplantation setting are ongoing to determine the potency and safety of combined retroviral expression of mbC46 and MGMT in relevant preclinical models. Successful conclusion of these studies will hopefully result in a phase I clinical trial testing the concept of generating an HIV-resistant autologous hematopoiesis.

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Hematopoiesis in Regenerated Bone Marrow on the Hydroxyapatite Scaffold.

Blood ◽

10.1182/blood.v110.11.1419.1419 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1419-1419

Author(s):

Atsushi Fujita ◽

Makoto Migita ◽

Takahiro Ueda ◽

Yoshitaka Fukunaga ◽

Takashi Shimada

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Lentiviral Vector ◽

Wide Distribution ◽

Whole Body ◽

Hematopoietic Stem ◽

In Vivo Bioluminescence Imaging ◽

Hydroxyapatite Scaffold

Abstract Background: In bone marrow (BM), osteoblastic cells lining the endosteal surface are a key component of the niche to promote and regulate hematopoietic stem cells (HSCs). Anatomical architecture of BM could be regenerated by culturing BM derived stromal cells on the hydroxyapatite (HA) scaffold. In the present study, we examined whether the regenerated BM has the ability to support HSCs in vivo. Methods: Our study was designed as follows; BM stromal cells from C57/BL6 mice (Ly5.2) were cultured on the HA scaffold with numerous small pores for 3 days in vitro and the scaffold with attached cells was implanted subcutaneously onto the back of C57/BL6 recipient mice. 4.0x105 Lineage negative (Lin−) Ly5.1 BM cells transduced with a lentiviral vector containing the luciferase (Luc) gene were intravenously administered into the recipient mice after lethal irradiation. Eight weeks after transplantation, the scaffolds were removed from the first recipient mice and subcutaneously transplanted into the lethally irradiated second recipient mice. The mice also received fresh Ly5.2 BM cells for survival. Biodistribution and kinetics of Luc+ Ly5.1 cells were monitored by in vivo bioluminescence imaging and FACS. Results: In the secondary transplanted mice, Luc+ hematopoitic cells were detected in the scaffolds for at least 6 months after transplantation. Subcutaneous injection of G-CSF resulted in wide distribution of bioluminescence signals from the original scaffolds to whole body including the head, extremities, chest, and abdomen. The presence of Ly5.1 B and T lymphocytes in the circulation was confirmed by FACS analysis 5 months after secondary transplantation. Conclusions: The regenerated BM on the HA scaffold is capable of supporting HSCs in vivo suggesting that the functional niche is reconstituted. Hematopoiesis in the regenerated BM may have a significant impact for development of new therapeutic strategies for various hematopoietic diseases.

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The Bone Marrow "Mystery Population" of Stem Cells 20 Years Later - a Puzzle Solved?

Blood ◽

10.1182/blood.v126.23.2392.2392 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2392-2392

Author(s):

Malwina Suszynska ◽

Daniel Pedziwiatr ◽

Magdalena J Kucia ◽

Mariusz Z Ratajczak ◽

Janina Ratajczak

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Receptor Expression ◽

Stem Cell Population ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Low Levels

Abstract Background . Almost 20 years ago, a "mystery" population of small stem cells with many of the phenotypic characteristics attributed to resting hematopoietic stem cells was identified in murine bone marrow (BM) (Stem Cells 1998, 16, 38-48). These cells expressed high levels of Sca-1, H-2K, and CD38 and low levels of Thy-1.1; they expressed CD45 antigen but were lineage-negative (lin-) for other hematopoietic markers. These cells incorporated only low levels of Rh123 and were resistant to the cytotoxic effects of 5-fluorouracil. The only phenotypic characteristic that distinguishes these cells from Sca-1+, Lin-, CD45+ Thy-1.1low long-term-reconstituting hematopoietic stem cell population is the lack of c-kit expression. In sum, this "mystery" population of small Sca-1+, lin-, c-kit- but CD45+ stem cells do not respond to hematopoietic growth factors in vitro, form in vivo spleen colonies, or reconstitute lethally irradiated mice. With our discovery of Sca-1+ Lin- CD45- very small embryonic-like stem cells (VSELs) in murine bone marrow (BM) (Leukemia 2006, 20, 857-869), we became interested in this "mystery" population of stem cells. VSELs, like the "mystery" population, are c-kit - and, if freshly isolated from BM, do not show any hematopoietic activity in standard in vitro and in vivo assays. In order to become specified to hematopoiesis, they need to be expanded over an OP-9 stromal support (Exp Hematol 2011;39:225-237). Hypothesis. Since (1) very small CD45- VSELs can be specified in OP-9 co-cultures into long-term reconstituting CD45+ HSCs, (2) the size of the "mystery" population is intermediate between VSELs and HSCs, and (3) VSELs and HSCs differ in cell surface receptor expression, we hypothesized that the "mystery" population is a missing developmental intermediate between VSELs and HSCs. Materials and Methods . Multicolor FACS analysis was employed to compare size and expression of surface markers between murine BM HSCs, the unknown population of stem cells, and VSELs. Next, the populations of small Sca-1+ H2-K+ lin- c-kit+ CD38+/- CD45+ cells (HSCs), smaller Sca-1+ H-2K+ lin- c-kit- CD38+ CD45+ cells (the "mystery" population), and very small in size Sca-1+ H-2K+ lin- c-kit- CD38+/- CD45- cells (VSELs) were purified by FACS from BM (Figure 1) and tested for in vitro colony formation. All these cell populations were primed/expanded over OP-9 support and subsequently evaluated for their hematopoietic potential after passaging in consecutive methylocellulose cultures (passages 1-4). RQ-PCR analysis was employed for detection of pluripotency marker expression as well as hematopoietic gene expression. Results . We found that, in contrast to HSCs, neither freshly sorted stem cells from the "mystery" BM population nor, as expected, VSELs grew hematopoietic colonies in standard methylcellulose cultures. This was also an important step in excluding contamination of our sorted populations with clonogenic cells. We also found that, while VSELs highly expressed Oct-4, this transcription factor was expressed at very low levels in the "mystery" population and was not detectable in HSCs. The most important observation was that the "mystery" population of stem cells became specified in OP-9-supported cultures into clonogenic HSPCs, and this specification occurred faster than the delayed specification of VSELs. VSELs first became enriched for HSPCs after acquiring CD45 antigen expression. Conclusions . Based on the results presented, we propose that the "mystery" population in murine BM is a population of stem cells intermediate between the most primitive population of BM-residing stem cells (VSELs) and the population of stem cells already specified to lympho-hematopoietic development (HSCs). Disclosures No relevant conflicts of interest to declare.

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HOXB6 overexpression in murine bone marrow immortalizes a myelomonocytic precursor in vitro and causes hematopoietic stem cell expansion and acute myeloid leukemia in vivo

Blood ◽

10.1182/blood-2004-04-1583 ◽

2005 ◽

Vol 105 (4) ◽

pp. 1456-1466 ◽

Cited By ~ 82

Author(s):

Neal A. Fischbach ◽

Sofia Rozenfeld ◽

Weifang Shen ◽

Stephen Fong ◽

Daniel Chrobak ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Hox Genes ◽

Cooperative Interaction ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Acute Myeloid

AbstractThe HOX family of homeobox genes plays an important role in normal and malignant hematopoiesis. Dysregulated HOX gene expression profoundly effects the proliferation and differentiation of hematopoietic stem cells (HSCs) and committed progenitors, and aberrant activation of HOX genes is a common event in human myeloid leukemia. HOXB6 is frequently overexpressed in human acute myeloid leukemia (AML). To gain further insight into the role of HOXB6 in hematopoiesis, we overexpressed HOXB6 in murine bone marrow using retrovirus-mediated gene transfer. We also explored structure-function relationships using mutant HOXB6 proteins unable to bind to DNA or a key HOX-binding partner, pre–B-cell leukemia transcription factor-1 (PBX1). Additionally, we investigated the potential cooperative interaction with myeloid ecotropic viral integration site 1 homolog (MEIS1). In vivo, HOXB6 expanded HSCs and myeloid precursors while inhibiting erythropoiesis and lymphopoiesis. Overexpression of HOXB6 resulted in AML with a median latency of 223 days. Coexpression of MEIS1 dramatically shortened the onset of AML. Cytogenetic analysis of a subset of HOXB6-induced AMLs revealed recurrent deletions of chromosome bands 2D-E4, a region frequently deleted in HOXA9-induced AMLs. In vitro, HOXB6 immortalized a factor-dependent myelomonocytic precursor capable of granulocytic and monocytic differentiation. These biologic effects of HOXB6 were largely dependent on DNA binding but independent of direct interaction with PBX1.

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Hematopoietic overexpression of the transcription factor Erg induces lymphoid and erythro-megakaryocytic leukemia

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1213454109 ◽

2012 ◽

Vol 109 (38) ◽

pp. 15437-15442 ◽

Cited By ~ 29

Author(s):

Catherine L. Carmichael ◽

Donald Metcalf ◽

Katya J. Henley ◽

Elizabeth A. Kruse ◽

Ladina Di Rago ◽

...

Keyword(s):

Bone Marrow ◽

Transcription Factor ◽

Cell Function ◽

Leukemic Cell ◽

Lymphoid Leukemia ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Cell Clones

The transcription factor encoded by the E-twenty-six (ETS)-related gene, ERG, is an essential regulator of hematopoietic stem cell function and a potent human oncoprotein. Enforced expression of ERG in murine hematopoietic cells leads to the development of a well-characterized lymphoid leukemia and a less well-defined non lymphoid disease. To clarify the latter, we generated murine bone marrow chimeras with enforced Erg expression in engrafted hematopoietic progenitor cells. As expected, these mice developed lymphoid leukemia. However, the previously reported non lymphoid disease that developed was shown to be a uniform, transplantable leukemia with both erythroid and megakaryocytic characteristics. In vivo, this disease had the overall appearance of an erythroleukemia, with an accumulation of immature erythroblasts that infiltrated the bone marrow, spleen, liver, and lung. However, when stimulated in vitro, leukemic cell clones exhibited both erythroid and megakaryocytic differentiation, suggesting that transformation occurred in a bipotential progenitor. Thus, in mice, Erg overexpression induces the development of not only lymphoid leukemia but also erythro-megakaryocytic leukemia.

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Mesenchymal stromal cell remodeling of a gelatin hydrogel microenvironment defines an artificial hematopoietic stem cell niche

10.1101/289553 ◽

2018 ◽

Author(s):

Aidan E. Gilchrist ◽

Sunho Lee ◽

Yuhang Hu ◽

Brendan A.C. Harley

Keyword(s):

Cell Culture ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Cell Activity ◽

Matrix Remodeling ◽

Hematopoietic Stem ◽

Murine Bone Marrow

AbstractHematopoietic stem cells (HSCs) reside in the bone marrow within discrete niches defined by a complex milieu of external signals including biophysical cues, bound and diffusible biomolecules, and heterotypic cell-cell interactions. Recent studies have shown the importance of autocrine-mediated feedback of cell-secreted signals and the interplay between matrix architecture and biochemical diffusion on hematopoietic stem cell activity. Autocrine and paracrine signaling from HSCs and niche-associated mesenchymal stromal cells (MSCs) have both been suggested to support HSC maintenance in vivo and in vitro. Here we report the development of a library of methacrylamide-functionalized gelatin (GelMA) hydrogels to explore the balance between autocrine feedback and paracrine signals from co-encapsulated murine bone marrow MSCs on murine HSCs. The use of a degradable GelMA hydrogel enables the possibility for significant MSC-mediated remodeling, yielding dynamic shifts in the matrix environment surrounding HSCs. We identify a combination of an initially low-diffusivity hydrogel and a 1:1 HSPC:MSC seeding ratio as conducive to enhanced HSC population maintenance and quiescence. Further, gene expression and serial mechanical testing data suggests that MSC-mediated matrix remodeling is significant for the long-term HSC culture, reducing HSC autocrine feedback and potentially enhancing MSC-mediated signaling over 7-day culture in vitro. This work demonstrates the design of an HSC culture system that couples initial hydrogel properties, MSC co-culture, and concepts of dynamic reciprocity mediated by MSC remodeling to achieve enhanced HSC maintenance.One Sentence SummaryCoupling effects of hydrogel biotransport, heterotypic cell culture, and matrix remodeling enhances hematopoietic stem cell culture and quiescence.

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A seleno-hormetine protects bone marrow hematopoietic cells against ionizing radiation-induced toxicity

10.1101/432286 ◽

2018 ◽

Author(s):

Bartolini Desirée ◽

Wang Yanzhong ◽

Zhang Jie ◽

Giustarini Daniela ◽

Rossi Ranieri ◽

...

Keyword(s):

Bone Marrow ◽

Ionizing Radiation ◽

Preclinical Development ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Major Player ◽

Stem And Progenitor Cells ◽

Thiol Peroxidase

Abstract2,2’-diselenyldibenzoic acid (DSBA) is a mild thiol peroxidase agent presently in preclinical development. This study reports that the drug has novel seleno-hormetic properties in both murine bone marrow and human liver cells. According with previous in vitro findings, mechanistic aspects of such properties were confirmed to include the activation of Nrf2 transcription factor and an increased expression of downstream stress response genes in the liver and in hematopoietic stem and progenitor cells of the myeloid lineage. These genes include glutathione S-transferase that is reported to represent a major player in the metabolism and pharmacological function of seleno-organic compounds. As a practical application, DSBA administration prevented bone marrow toxicities following acute exposure to sub-lethal doses of ionizing radiation in C57 BL/6 mice.In conclusion, this study demonstrates for the first time the pharmacological properties of DSBAin vivo. The findings suggest applications for this selenohormetine in radioprotection and prevention protocols.

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Reciprocal expression of CD38 and CD34 by adult murine hematopoietic stem cells

Blood ◽

10.1182/blood.v97.9.2618 ◽

2001 ◽

Vol 97 (9) ◽

pp. 2618-2624 ◽

Cited By ~ 52

Author(s):

Fumihito Tajima ◽

Takao Deguchi ◽

Joseph H. Laver ◽

Haiqun Zeng ◽

Makio Ogawa

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Steady State ◽

Adult Stem Cells ◽

Human Stem Cells ◽

Hematopoietic Stem ◽

Reciprocal Expression

Abstract The effects of activation of adult murine stem cells on their expression of CD38 were studied using a murine transplantation model. First, the published finding that the majority of long-term engrafting cells from normal adult steady-state marrow are CD38+ was confirmed. Next, it was determined that the majority of stem cells activated in vivo by injection of 5-fluorouracil (5-FU) or mobilized by granulocyte colony-stimulating factor are CD38−. Stem cells that were activated in culture with interleukin-11 and steel factor were also CD38−. Previous studies have shown that expression of CD34 by adult stem cells is also modulated by in vivo or in vitro activation. To determine whether there is reciprocal expression of CD38 and CD34, 4 populations of post–5-FU marrow cells were analyzed. The majority of the stem cells were in the CD38−CD34+ fraction. However, secondary transplantation experiments indicated that when the bone marrow reaches steady state, the majority of the stem cells become CD38+CD34−. In addition, the minority populations of CD34+ stem cells that occur in steady-state bone marrow are CD38−. This reversible and reciprocal expression of CD38 and CD34 by murine stem cells may have implications for the phenotypes of human stem cells.

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Transcriptional Targeting of Retroviral Vectors to the Erythroblastic Progeny of Transduced Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v93.10.3276.410k08_3276_3285 ◽

1999 ◽

Vol 93 (10) ◽

pp. 3276-3285 ◽

Cited By ~ 49

Author(s):

Alexis Grande ◽

Bianca Piovani ◽

Alessandro Aiuti ◽

Sergio Ottolenghi ◽

Fulvio Mavilio ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Retroviral Vectors ◽

Transcription Unit ◽

Hematopoietic Stem ◽

Gene Therapy Vector ◽

Murine Bone Marrow

Targeted expression to specific tissues or cell lineages is a necessary feature of a gene therapy vector for many clinical applications, such as correction of hemoglobinopathies or thalassemias by transplantation of genetically modified hematopoietic stem cells. We developed retroviral vectors in which the constitutive viral enhancer in the U3 region of the 3′ LTR is replaced by an autoregulatory enhancer of the erythroid-specific GATA-1 transcription factor gene. The replaced enhancer is propagated to the 5′ LTR upon integration into the target cell genome. The modified vectors were used to transduce human hematopoietic cell lines, cord blood-derived CD34+ stem/progenitor cells, and murine bone marrow repopulating stem cells. The expression of appropriate reporter genes (▵LNGFR, EGFP) was analyzed in the differentiated progeny of transduced stem cells in vitro, in liquid culture as well as in clonogenic assay, and in vivo, after bone marrow transplantation in lethally irradiated mice. The GATA-1 autoregulatory enhancer effectively restricts the expression of the LTR-driven proviral transcription unit to the erythroblastic progeny of both human progenitors and mouse-repopulating stem cells. Packaging of viral particles, integration into the target genome, and stability of the integrated provirus are not affected by the LTR modification. Enhancer replacement is therefore an effective strategy to target expression of a retroviral transgene to a specific progeny of transduced hematopoietic stem cells.

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Identification of a Novel Murine CD45 Negative Bone Marrow-Derived Cell Population with In Vivo Marrow Repopulating Potential Following Hematopoietic Specification In Vitro.

Blood ◽

10.1182/blood.v106.11.1719.1719 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1719-1719

Author(s):

Edward F. Srour ◽

Tamara L. Horvath

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Pcr Analysis ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Murine Bone Marrow

Abstract Murine bone marrow-derived cells expressing Sca-1+c-kit+lin− (KSL), as well as subfractions of these cells, represent an enriched population of hematopoietic stem cells (HSC) capable of long-term reconstitution of lethally irradiated recipients. Commitment to the hematopoietic lineage is invariably associated with expression of the pan-leukocyte marker CD45 which is also expressed on KSL cells. Whether KSL cells are the most primitive population of HSC present in the bone marrow (BM) is not fully resolved. We hypothesized that putative HSC that are more primitive than KSL cells may not express CD45 or genetic elements that mark early hematopoietic specification and commitment, but may mature under appropriate conditions into CD45+ cells capable of hematopoietic differentiation in conditioned hosts. BM cells from 8 to 10-week old BoyJ mice were collected by flushing and erythrocytes were lysed. The remaining cells were stained and sorted to yield CD45+ Sca-1+ c-kit+ (CD45+HSC) and CD45− Sca-1+ c-kit− (CD45−) cells which represented approximately 0.02% of total cells analyzed. PCR analysis of both cell populations revealed that CD45+HSC expressed CD45 and SCL but not PU.1 while CD45− cells did not express any of these genes. Directly after sorting, CD45+HSC, but not CD45− cells contained clonogenic cells that gave rise to hematopoietic colonies in progenitor cell assays. Similarly, while fresh CD45+HSC were able to respond to exogenous hematopoietic cytokines including SCF, TPO, and FL in liquid suspension cultures as evidenced by expansion and differentiation, their CD45− counterparts failed to proliferate under these conditions and none survived beyond 7 days of culture. When transplanted competitively into lethally irradiated congenic recipients, only freshly isolated CD45+HSC sustained donor-derived hematopoiesis, whereas hematopoiesis in mice injected with freshly isolated CD45− cells was sustained long term by competitor cells and endogenous host-derived stem cells. Both groups of CD45+HSC and CD45− cells could be expanded on irradiated M210B4 stromal cells when supplemented with SCF, TPO, and FL, with CD45− cells giving rise to cobblestone foci of small, round translucent cells beginning on day 7 of culture. Cultured CD45+HSC continued to express CD45 and SCL and, depending on the length of culture, also expressed PU.1. Interestingly, after 15 days in culture, CD45− cells expressed CD45 by RT-PCR and FACS (in addition to Sca-1) and also expressed mRNA for SCL. Given the ability of CD45− cells to expand under these conditions and to acquire CD45 expression, we next compared the repopulating potential of fresh and cultured CD45+HSC and CD45− cells using lethally irradiated C57Bl/6 recipients. As expected, fresh CD45+HSC sustained donor-derived engraftment and culture of these cells over M210B4 for 15 days reduced their repopulating potential more than 7-fold. In contrast, CD45− cells maintained on M210B4 (the expansion equivalent of 750 cells seeded) contributed to hematopoietic engraftment, albeit at low levels (under 5% chimerism). These data demonstrate that CD45− Sca-1+ c-kit− cells may be marrow resident precursors of hematopoietic stem cells and suggest that early stages of the HSC hierarchy may include CD45− cells. Whether these CD45− cells also posses endothelial differentiation potential and can give rise to CD45+HSC in vivo is now under investigation.

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