The Roles of Pten in Hematopoietic Stem Cell Regulation and Leukemogenesis.

Abstract Pten was the first phosphatase identified as a tumor suppressor and one of the most frequently mutated genes involved in human tumor/cancer. Pten, involved in regulation of both PI3K/Akt and MEK/Erk activity, is downstream of growth factor, cytokine, integrin and cadherin signaling pathways and therefore plays important roles in cell growth, survival, differentiation, metabolism and migration. Although Pten mutation is not common in leukemic cells, phosphorylated Pten (p-Pten), which represents the inactive form of Pten, has been observed in a majority of acute myeloid leukemias that are associated with poor clinical outcomes. To explore the role of Pten in hematopoietic stem cell (HSC) regulation and leukemogenesis, we generated an interferon-inducible Pten knockout mouse by crossing Mx1Cre mice with Ptenloxp mice. All of the mutant mice developed myeloproliferative disorder characterized by increased peripheral white blood cell counts, hyperproliferative macrophages and granulocytes in bone marrow and spleen, and multiple tissue infiltration by myeloid cells. The HSC number was decreased in the bone marrow but mobilized and expanded in the spleen. Extra-medullar hematopoiesis was shown by dramatically increased myeloid and erythroid progenitors in the spleen. B lymphocyte differentiation was blocked at the common lymphoid progenitor stage, while the T cell number was increased in all hematopoietic tissues. Compared to wild type, Pten mutant HSCs and progenitor cells were highly proliferative, forming larger colonies in an in vitro culture study. However, Pten mutant HSCs showed reduced competency in repopulation assay after in vivo bone marrow transplantation. Our study demonstrates that Pten plays important roles in restricting HSC activation, proliferation and mobilization. Pten also plays a role in HSC lineage decision by favoring myeloid differentiation at the expense of B lymphocyte lineage.

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Bone Marrow Cell Aggregates: A Way of Collecting the Adult Human Hematopoietic Stem Cell Niche

Blood ◽

10.1182/blood.v124.21.4363.4363 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4363-4363

Author(s):

Alexandre Janel ◽

Nathalie Boiret-Dupré ◽

Juliette Berger ◽

Céline Bourgne ◽

Richard Lemal ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Confocal Microscopy ◽

Hematopoietic Stem Cell ◽

Biological Samples ◽

Mesenchymal Cells ◽

Hematopoietic Stem ◽

Bm Niche ◽

Cd34 Cells

Abstract Hematopoietic stem cell (HSC) function is critical in maintaining hematopoiesis continuously throughout the lifespan of an organism and any change in their ability to self-renew and/or to differentiate into blood cell lineages induces severe diseases. Postnatally, HSC are mainly located in bone marrow where their stem cell fate is regulated through a complex network of local influences, thought to be concentrated in the bone marrow (BM) niche. Despite more than 30 years of research, the precise location of the HSC niche in human BM remains unclear because most observations were obtained from mice models. BM harvesting collects macroscopic coherent tissue aggregates in a cell suspension variably diluted with blood. The qualitative interest of these tissue aggregates, termed hematons, was already reported (first by I. Blaszek's group (Blaszek et al., 1988, 1990) and by our group (Boiret et al., 2003)) yet they remain largely unknown. Should hematons really be seen as elementary BM units, they must accommodate hematopoietic niches and must be a complete ex vivo surrogate of BM tissue. In this study, we analyzed hematons as single tissue structures. Biological samples were collected from i) healthy donor bone marrow (n= 8); ii) either biological samples collected for routine analysis by selecting bone marrow with normal analysis results (n=5); or iii) from spongy bone collected from the femoral head during hip arthroplasty (n=4). After isolation of hematons, we worked at single level, we used immunohistochemistry techniques, scanning electronic microscopy, confocal microscopy, flow cytometry and cell culture. Each hematon constitutes a miniature BM structure organized in lobular form around the vascular tree. Hematons are organized structures, supported by a network of cells with numerous cytoplasmic expansions associated with an amorphous structure corresponding to the extracellular matrix. Most of the adipocytes are located on the periphery, and hematopoietic cells can be observed as retained within the mesenchymal network. Although there is a degree of inter-donor variability in the cellular contents of hematons (on average 73 +/- 10 x103 cells per hematon), we observed precursors of all cell lines in each structure. We detected a higher frequency of CD34+ cells than in filtered bone marrow, representing on average 3% and 1% respectively (p<0.01). Also, each hematon contains CFU-GM, BFU-E, CFU-Mk and CFU-F cells. Mesenchymal cells are located mainly on the periphery and seem to participate in supporting the structure. The majority of mesenchymal cells isolated from hematons (21/24) sustain in vitro hematopoiesis. Interestingly, more than 90% of the hematons studied contained LTC-ICs. Furthermore, when studied using confocal microscopy, a co-localization of CD34+ cells with STRO1+ mesenchymal cells was frequently observed (75% under 10 µm of the nearest STRO-1+ cell, association statistically highly significant; p <1.10-16). These results indicate the presence of one or several stem cell niches housing highly primitive progenitor cells. We are confirming these in vitro data with an in vivo xenotransplantation model. These structures represent the elementary functional units of adult hematopoietic tissue and are a particularly attractive model for studying homeostasis of the BM niche and the pathological changes occurring during disease. Disclosures No relevant conflicts of interest to declare.

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Abstract 156: Angiotensin Ii Regulates Hematopoietic Stem Cell Proliferation, Differentiation, and Engraftment Efficacy

Hypertension ◽

10.1161/hyp.62.suppl_1.a156 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Seungbum Kim ◽

Edward W Scott ◽

Mohan K Raizada

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Angiotensin Ii ◽

Hematopoietic Stem Cell ◽

Peripheral Blood ◽

Ang Ii ◽

Hematopoietic Stem ◽

Proliferation And Differentiation ◽

Important Regulator

INTRODUCTION: Emerging evidence indicates that differentiation and mobilization of hematopoietic stem cell (HSC) are critical in the development and establishment of hypertension-linked vascular pathophysiology. This, coupled with the intimate involvement of a hyperactive renin-angiontensin system in hypertension, led us to propose the hypothesis that chronic angiotensin II (Ang II) infusion would regulate HSC proliferation and differentiation at the bone marrow level. METHODS: 1) Ang II was chronically infused into C57BL6 mice using mini-osmotic pumps (1500ng/kg/min) for 3 weeks. This resulted in an increase in MAP of 45mmHg. Bone marrow, peripheral blood and splenocytes from control and Ang II-treated mice were analyzed using FACS. 2) 0.5-3 X10 4 GFP + Sca-1 + , c-Kit + , Lin - (SKL) HSC were pre-incubated with Ang II for 24h in vitro (100μg/ml), rinsed and injected into lethally irradiated C57BL6 mice. Donor derived GFP + cells were analyzed by FACS and histology to evaluate engraftment efficiency. RESULTS: We observed a 32% decrease of HSCs in the bone marrow of Ang II treated mice. In addition, there was an 29-52% increase in the number of CX3CR1+/Gr-1- monocyte in the peripheral blood and spleen. These changes in HSC and myeloid cells were blocked by co-treatment of Losartan (60mg/kg/day, ip injection). Next, we investigated if Ang II affects HSC homing and engraftment efficacy, which are critical steps in successful bone marrow transplantation. We observed a significant delay of the homing GFP+ SKL cells that were pre-treated with Ang II in lethally irradiated recipient mice. In addition, the SKL cells treated with Ang II failed to efficiently engraft to the innate osteoblastic niche. Consistent with this observation, colony formation unit-Spleen (CFU-S) in the Ang II infused recipients was reduced to 65% compared to control mice. CONCLUSION: These observations demonstrate that hypertension induced by chronic Ang II infusion significantly impairs the engraftment ability of HSC in the bone marrow, which appears to be mediated by the AT1R on HSC and that Ang II accelerates HSC differentiation into myeloid lineage. These multifaceted roles of Ang II indicate that Ang II acts as an important regulator of HSC in the bone marrow.

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Culture from human bone marrow of blast progenitor cells with an extensive proliferative capacity

Blood ◽

10.1182/blood.v69.3.804.804 ◽

1987 ◽

Vol 69 (3) ◽

pp. 804-808 ◽

Cited By ~ 39

Author(s):

SD Rowley ◽

SJ Sharkis ◽

C Hattenburg ◽

LL Sensenbrenner

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Human Bone ◽

Human Bone Marrow ◽

Vitro Assay ◽

Hematopoietic Stem ◽

Blast Cells ◽

Normal Human

Abstract The investigation of human hematopoiesis is limited by the lack of an in vitro assay for the most primitive hematopoietic stem cell. In this report, we describe the culture from normal human bone marrow of unique colonies of morphologically immature cells with scanty, agranular, cytoplasm and a primitive nucleus with nucleoli. These “blast” cells demonstrate a significant ability for the generation of secondary colonies of multiple lineages, including additional blast cell colonies. These colonies are detected at various times during the culture period of up to 28 days. Neither the time of appearance in primary culture nor any feature of the morphological appearance of the blast cells is correlated with replating ability or the differentiation pathway followed. The progenitor cell giving rise to these colonies may represent the earliest pluripotent hematopoietic stem cell yet grown in culture.

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Ontogeny of hematopoietic stem cell development: reciprocal expression of CD33 and a novel molecule by maturing myeloid and erythroid progenitors

Blood ◽

10.1182/blood.v82.3.792.bloodjournal823792 ◽

1993 ◽

Vol 82 (3) ◽

pp. 792-799

Author(s):

C Brashem-Stein ◽

DA Flowers ◽

FO Smith ◽

SJ Staats ◽

RG Andrews ◽

...

Keyword(s):

Monoclonal Antibody ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Bone Marrow Culture ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Selective Expression ◽

Reciprocal Expression

We have identified a molecule expressed by human marrow granulocyte/monocyte colony-forming cells (CFU-GM), erythroid colony- forming cells (CFU-E), and erythroid burst-forming units (BFU-E), but not their precursors detectable in long-term bone marrow culture. This antigen, detected by flow microfluorimetry using monoclonal antibody 7B9, is coexpressed with CD33 on many CD34+ CFCs, although only the 7B9 antigen was detected on a portion of BFU-E and CFU-E, whereas only CD33 was found on a portion of CFU-GM. Antibody 7B9 appears to be useful for isolating subsets of progenitors based on their common or selective expression of 7B9 antigen and CD33.

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Etoposide-mediated interleukin-8 secretion from bone marrow stromal cells induces hematopoietic stem cell mobilization

10.21203/rs.2.22219/v1 ◽

2020 ◽

Author(s):

Ka-Won Kang ◽

Seung-Jin Lee ◽

Ji Hye Kim ◽

Byung-Hyun Lee ◽

Seok Jin Kim ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Hematopoietic Stem ◽

Cell Counts ◽

Cd34 Cells

Abstract Background This study assessed the mechanism of hematopoietic stem cell (HSC) mobilization using etoposide with granulocyte-colony stimulating factor (G-CSF) and determined how it differed from that using cyclophosphamide with G-CSF or G-CSF alone.Methods The study analyzed data from 173 non-Hodgkin’s lymphoma patients who underwent autologous peripheral blood stem cell transplantation (auto-PBSCT), in vitro experiments using HSCs and bone marrow stromal cells (BMSCs), and in vivo mouse model studies.Results The etoposide with G-CSF mobilization group showed the highest yield of CD34+ cells and the lowest change in white blood cell counts during mobilization. Etoposide triggered interleukin (IL)-8 secretion from BMSCs and caused long-term BMSC toxicity, which were not observed with cyclophosphamide treatment. The expansion of CD34+ cells cultured in BMSC-conditioned medium containing IL-8 was more remarkable than that without IL-8. The expression of CXCR2, mTOR, and cMYC in HSCs was gradually enhanced at 1, 6, and 24 h after IL-8 stimulation. In animal studies, the etoposide with G-CSF mobilization group presented stronger expression of IL-8-related cytokines and MMP9 and scantier expression of SDF-1 in the bone marrow, compared to the other groups not treated with etoposide.Conclusion Collectively, the unique mechanism of etoposide with G-CSF-mediated mobilization is associated with the secretion of IL-8 from BMSCs, causing the enhanced proliferation and mobilization of HSCs in the bone marrow, which was not observed in the mobilization using cyclophosphamide with G-CSF or G-CSF alone. Moreover, the long-term toxicity of etoposide to BMSC emphasizes the need for further studies to develop more efficient and safe chemo-mobilization strategies.

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Hematopoietic Stem Cell Expansion by HOXB4 Is Greatly Enhanced in p21 Deficient Stem Cells.

Blood ◽

10.1182/blood.v104.11.1688.1688 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1688-1688 ◽

Cited By ~ 1

Author(s):

Noriko Miyake ◽

Ann C.M. Brun ◽

Mattias Magnusson ◽

David T. Scadden ◽

Stefan Karlsson

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, enforced expression of HOXB4 is a potent stimulus for murine hematopoietic stem cell (HSC) self-renewal. Murine HSCs engineered to overexpress HoxB4 expand significantly more than control cells in vivo and ex vivo while maintaining a normal differentiation program. HSCs are regulated by the cell proliferation machinery that is intrinsically controlled by cyclin-dependent kinase inhibitors such as p21Cip1/Waf1(p21) and p27Kip1 (p27). The p21 protein restricts cell cycling of the hematopoietic stem cell pool and maintains hematopoietic stem cell quiescence. In order to ask whether enhanced proliferation due to HOXB4 overexpression is mediated through suppression of p21 we overexpressed HOXB4 in hematopoietic cells from p21−/− mice. First, we investigated whether human HOXB4 enhances in vitro expansion of BM cells from p21−/− mice compared to p21+/+ mice. 5FU treated BM cells from p21−/− or p21+/+ mice were pre-stimulated with SCF, IL-6, IL-3 for 2 days followed by transduction using retroviral vector expressing HOXB4 together with GFP (MIGB4) or the control GFP vector (MIG). The cells were maintained in suspension cultures for 13 days and analyzed for GFP positive cells by flow-cytometry. Compared to MIG transduced BM cells from p21+/+ mice (MIG/p21+), the numbers of GFP positive cells were increased 1.1-fold in MIG/p21−, 3.2-fold in MIGB4/p21+ and 10.0-fold in MIGB4/p21− respectively (n=5). CFU assays were performed after 13 days of culture. The numbers of CFU were increased 4.8-fold in MIG/p21−, 19.5-fold in MIG/p21+ and 33.9 -fold in MIGB4/p21− respectively. Next, we evaluated level of HSCs expansion by bone marrow repopulation assays. After 12-days of culture, 1.5 x 105 MIGB4 or MIG-transduced cells (Ly5.2) were transplanted into lethally irradiated mice in combination with 8 x 105 fresh Ly5.1 bone marrow cells. Sixteen weeks after transplantation, no Ly5.2 cells could be detected in MIG/p21+ or MIG/p21− transplanted mice (n=6). In contrast, Ly5.2 positive cells were detected in both MIGB4/p21+/+ and MIGB4/p21−/− cells. The % of Ly5.2 positive cells in MIGB4/p21− transplanted mice was 9.9-fold higher than MIGB4/p21+ transplanted mice. (38.4 % vs 3.9 %, P<0.02, n=5). These Ly5.2 positive cells differentiated into all lineages, as determined by proportions of Mac-1, B-220, CD3 and Ter119 positive populations. Currently, we are enumerating the expansion of HOXB4 transduced HSCs in p21 deficient BM cells using the CRU assay. Our findings suggest that HOXB4 increases the self-renewal of hematopoietic stem cells by a mechanism that is independent of p21. In addition, the findings demonstrate that deficiency of p21 in combination with enforced expression of HOXB4 can be used to rapidly and effectively expand hematopoietic stem cells.

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Host CD4+CD25+FoxP3+ Regulatory T Cells Facilitate Donor Engraftment Without Impacting Graft Versus Host Disease Onset After Major-Mismatched Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v122.21.1996.1996 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1996-1996

Author(s):

Antonio Pierini ◽

Hidekazu Nishikii ◽

Mareike Florek ◽

Dennis B Leveson-Gower ◽

Yuqiong Pan ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Cellular Therapy ◽

Hematopoietic Stem ◽

Graft Versus Host ◽

Donor Chimerism

Abstract A major challenge following allogeneic hematopoietic stem cell transplantation (HCT) is to establish persistent engraftment of donor hematopoietic cells. Many strategies have been developed to permit engraftment involving high dose chemotherapy, serotherapy with anti-lymphocyte drugs or myeloablative irradiation resulting in highly toxic conditioning regimens. The introduction of less harmful therapies could result in less toxicity especially in the major mismatched setting and when reduced intensity conditioning is required. While recent studies have explored the mechanisms through which donor-type CD4+CD25+FoxP3+ regulatory T cells (Tregs) restrict the development of graft versus host and host versus graft reactions, less is known about the role of host-type Treg in the transplant setting. In syngeneic and minor mismatched HCT host Tregs comprise a major component of the Treg compartment in the first weeks after transplant. Moreover the transplant of in vitro primed host Tregs can improve donor engraftment in major mismatched models of HCT; therefore host Tregs could be one of the key controllers of the host versus graft reaction mediated by residual host CD4+ and CD8+ conventional T cells (Tcons), possibly influencing graft versus host disease (GvHD) onset and severity. In this study we investigated the role of host Treg after major mismatched HCT to understand their impact in graft facilitation and rejection and in GvHD induction and prevention. We investigated the mechanism through which this cell population works and we explored the feasibility and the effectiveness of host Treg adoptive transfer for cellular therapy in HCT animal models. Results CD4+CD25+FoxP3+ host Tregs persist for at least 28 days after total body irradiation (8 Gy) and transplantation of C57BL/6 (H-2b) T cell depleted bone marrow (TCD BM) into BALB/C (H-2d) mice. Host Treg could be found in spleen, lymph nodes and bone marrow with an increase in the Treg/CD4+ cell ratio. Moreover we observed that these residual host Tregs maintain their suppressive function in vitro if harvested 14 days after transplant and incubated with healthy mouse derived Tcons in a MLR. These results are even more relevant as transplanted mouse derived host Tcons lose their ability to proliferate confirming that host Tregs possess a numeric and functional advantage compared to residual host Tcons. Using FOXP3-DTR mice as hosts we observed that host Treg ablation results in reduced donor chimerism after major mismatched TCD BM transplant (p < 0.01, analysis performed 2 months after transplant). At the same time, the absence of host Tregs favors host CD4+ T cell persistence (p < 0.001) and delays B cell reconstitution (p < 0.001). Furthermore, we hypothesized that host Treg act as an immunological barrier for HSCs, providing a protective immunological niche. Confocal microscopic analysis of femurs performed at day 7 after TCD BM transplant confirmed that hypothesis showing host Tregs clustering in the epiphysis where donor hematopoietic stem cell (HSC) engraftment is mainly detectable. To strengthen these results and to provide a clinical translatable tool, we adoptively transferred 5x105/mouse highly purified unmanipulated host Tregs in a non myeloablative (TBI 5.5 Gy) major mismatched model of rejection. We found that the transferred host Tregs induce persistent full donor chimerism if injected together with a sublethal dose of donor Tcons (5x105/mouse, p=0.016) and transiently enhance donor chimerism in the first three weeks after transplant if injected with low dose interleukin-2 (IL-2, 50,000 IU bid for 7 days; p < 0.001) without impacting on GvHD incidence and lethality. The relatively low dose of injected Tregs, the possibility to stimulate and expand them in vivo with IL-2 and the safety of this model provide the first evidence of the feasibility of this clinical approach. Conclusion Our findings indicate that host Tregs facilitate bone marrow engraftment in major mismatched HCT models without impacting GvHD. Notably, our observations on the bone marrow environment after transplant strongly suggest that host Tregs can play a role in building the donor HSC cell niche. Finally host Treg adoptive transfer proved to be feasible and effective in animal models providing a new tool for cellular therapy and clinical translation. Disclosures: No relevant conflicts of interest to declare.

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Culture from human bone marrow of blast progenitor cells with an extensive proliferative capacity

Blood ◽

10.1182/blood.v69.3.804.bloodjournal693804 ◽

1987 ◽

Vol 69 (3) ◽

pp. 804-808 ◽

Cited By ~ 1

Author(s):

SD Rowley ◽

SJ Sharkis ◽

C Hattenburg ◽

LL Sensenbrenner

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Human Bone ◽

Human Bone Marrow ◽

Vitro Assay ◽

Hematopoietic Stem ◽

Blast Cells ◽

Normal Human

The investigation of human hematopoiesis is limited by the lack of an in vitro assay for the most primitive hematopoietic stem cell. In this report, we describe the culture from normal human bone marrow of unique colonies of morphologically immature cells with scanty, agranular, cytoplasm and a primitive nucleus with nucleoli. These “blast” cells demonstrate a significant ability for the generation of secondary colonies of multiple lineages, including additional blast cell colonies. These colonies are detected at various times during the culture period of up to 28 days. Neither the time of appearance in primary culture nor any feature of the morphological appearance of the blast cells is correlated with replating ability or the differentiation pathway followed. The progenitor cell giving rise to these colonies may represent the earliest pluripotent hematopoietic stem cell yet grown in culture.

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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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CD34+ Cells Derived from Human Embryonic Stem Cells Demonstrate Hematopoietic Stem Cell Potential In Vitro and in Vivo.

Blood ◽

10.1182/blood.v104.11.564.564 ◽

2004 ◽

Vol 104 (11) ◽

pp. 564-564 ◽

Cited By ~ 2

Author(s):

Dan S. Kaufman ◽

Petter S. Woll ◽

Colin H. Martin ◽

Jon L. Linehan ◽

Xinghui Tian

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Es Cells ◽

Es Cell ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Human Es Cells

Abstract We have previously described methods to use either stromal cell co-culture or embryoid-body (EB) formation to support the hematopoietic development of undifferentiated human ES cells (both H1 and H9 cell lines). Either FBS-based media or serum-free media with specific cytokines can be used to derive CD34+ cells, CD45+ cells and hematopoietic progenitors as identified by colony-forming cell (CFC) assays that give rise to mature myeloid, erythroid, and megakaryocytic cells. Genes such as RUNX1, HOXB4, TAL1, and GATA2, all known to be expressed during early hematopoiesis are up-regulated during hematopoietic differentiation of human ES cells. Here, we advance these studies to demonstrate that human ES cell-derived CD34+ cells function as early hematopoietic precursors in surrogate hematopoietic stem cell (HSC) assays. The long-term culture initiating cell (LTC-IC) assay is commonly used to quantify hematopoietic precursors that can be maintained in culture for 5 or more weeks. Human cord blood (CB)-derived CD34+ cells have a LTC-IC frequency of approximately 1:30. We demonstrate LTC-ICs can also be identified from human ES cell-derived CD34+ at a frequency of approximately 1:400. These results suggest CD34+ cells from human ES cells are more heterogeneous than CD34+ cells from CB. Furthermore, we now demonstrate in vitro culture of human ES cell-derived CD34+ cells identify these cells as lymphocyte precursors. Here, we used a natural killer (NK) cell-initiating cell assay (NK-IC) where CD34+ cells are cultured on AFT024 stromal cells in media containing IL15, IL7, and other defined cytokines for 2–4 weeks. Under these conditions, both CB and human ES cell-derived cells give rise to lymphoid cells (NK cells) with over 40% CD45+CD56+ cells. Under alternative culture conditions, CD3+ T cells can also be produced from CD34+ human ES cell-derived cells. Therefore, CD34+ cells derived from human ES cells represent both myeloid and lymphoid precursor cells. Since it is not possible to define a HSC population based solely on in vitro assays, we have examined the potential for human ES cell-derived hematopoietic cells to engraft in sublethally irradiated NOD/SCID mice. Detection of scid-repopulating cells (SRCs) are considered a better surrogate for HSCs. Bone marrow, peripheral blood, and splenocytes were examined for human CD34+ and CD45+ cells 3–6 months after injection of human ES cell-derived blood cells. PCR for human chromosome 17-specific alpha-satellite DNA was also done to confirm the presence of human cells in all mice showing evidence of engraftment. We consistently find stable engraftment with 0.5–3% human CD45+ cells in the bone marrow of these mice. To better define these cells as HSCs, secondary transplants also demonstrate stable engraftment. Importantly, no teratomas are demonstrated in mice injected with differentiated human ES cells. These results demonstrate that HSCs with long-term engraftment and multi-lineage potential can be routinely and efficiently generated from human ES cells.

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