The Role of BMP-4 in the Stromal-Maintenance of Haematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1709-1709
Author(s):  
Jonathon F. Hutton ◽  
Fiona Khor ◽  
Vlad Rozenkov ◽  
Ian D. Lewis ◽  
Richard J. D’Andrea
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Embryonic Stem ◽  
Human Cord Blood ◽  
Haematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Foetal Liver

Abstract Haematopoietic stem cells (HSCs) expand in number during fetal liver hematopoiesis via a process that is not understood. Establishment of conditions supporting HSC maintenance and expansion ex vivo is critical for wider application of cord blood derived HSC. We have shown that bone morphogenic protein (BMP) 4 secreted by mouse foetal liver cells contributes to expansion of cord blood-derived HSCs during in vitro co-culture. Significant levels of BMP-4 mRNA and secreted protein were produced by the supportive murine foetal liver stromal cell line AFT024. Supplementing 14 day co-cultures of AFT024 and human cord blood CD34+ cells with the BMP-4 antagonist Noggin, or a neutralising BMP-4 antibody decreased the proportion of cells maintaining a CD34+CD38−CD33− primitive phenotype (by 27.6% and 37.2% respectively), decreased CFU-GM expansion (by 20.6% and 22.2% respectively), and caused a large reduction in net expansion of long-term culture-initiating cells (LTC-IC) (by 31.7% and 61.5% respectively). The ability of BMP4 to support the multipotency and self-renewal of hematopoietic stem cells is consistent with a role recently shown on embryonic stem cells and suggests that it may act generally as a stem cell maintenance factor. Elucidating further the role of HSC growth factors such as BMP-4 in ex vivo culture may lead to development of defined systems for the routine clinical expansion of HSC.

Ex Vivo Treatment of Human Cord Blood with dmPGE2 Can Safely Increase the Potential for Hematopoietic Engraftment.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1190-1190
Author(s):  
Trista E. North ◽  
Wolfram Goessling ◽  
Myriam Armant ◽  
Grace S. Kao ◽  
Leslie E. Silberstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Relative Distribution ◽  
Human Cord Blood ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) are commonly used in transplantation therapy to rescue the hematopoietic and immune systems following systemic chemotherapy or irradiation. However, some patients receive inadequate numbers of HSCs and this often results in delayed reconstitution of hematopoiesis and immune function and associated toxicities. We previously demonstrated that a stabilized derivative of prostaglandin (PG) E2 increases vertebrate HSCs both in vivo and in vitro. 16,16-dimethyl PGE2 (dmPGE2) significantly increased HSCs during zebrafish embryogenesis and in the adult marrow following injury. Incubation of murine embryonic stem cells with dmPGE2 during embryoid body differentiation resulted in a dose-dependent increase in hematopoietic colonies, demonstrating that the function of PGE2 in HSC regulation is conserved in mammals. Finally, ex vivo treatment of murine bone marrow with dmPGE2 resulted in a 2-fold increase in engrafting cells in a limiting dilution competitive repopulation assay. No negative effects on serial transplantability of HSCs were observed in these animal models. To investigate the therapeutic potential of PGE2 for the amplification of blood stem cells, we exposed human cord blood (hCB) cells to dmPGE2 in vitro and measured the effects on stem and progenitor populations both in vitro and in vivo. Red cell depleted umbilical cord blood specimens, cryopreserved for clinical use, were thawed and divided for parallel processing. Ex vivo treatment of hCB cells for 1 hour with dmPGE2 in dextran/albumin had no negative impact on absolute cell count or the viability and relative distribution of both CD45 and CD34 positive cells compared to vehicle treated control hCB cells. Significantly, hCB treated with dmPGE2 produced enhanced numbers of GM and GEMM colonies in methylcellose CFU-C assays compared to controls. Human CB cells treated ex vivo with dmPGE2 for 1 hour and transplanted at a dose of 20 million live CD45+ cells per recipient were capable of repopulating NOD/SCID mice after sublethal irradiation. In comparative studies at 6 weeks post transplantation, human CD34+ and CD45+ cells could be detected in the marrow (>2%) of dmPGE2 treated (4/8) and control treated (1/6) recipients. Long-term and competitive transplantation experiments to assess the effect of dmPGE2 treatment on functional HSCs are currently in progress. Our data suggests that treatment of human cord blood products with dmPGE2 will be both safe and effective in achieving expansion of hematopoietic stem cells for transplantation in the clinical setting. TE North and W Goessling contributed equally to this work.

Ex vivo treatment of proliferating human cord blood stem cells with stroma-derived factor–1 enhances their ability to engraft NOD/SCID mice

Blood ◽  
2002 ◽  
Vol 99 (9) ◽  
pp. 3454-3457 ◽  
Author(s):  
Hanno Glimm ◽  
Patrick Tang ◽  
Ian Clark-Lewis ◽  
Christof von Kalle ◽  
Connie Eaves
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
Tgf Β1

Abstract Ex vivo proliferation of hematopoietic stem cells (HSCs) is important for cellular and gene therapy but is limited by the observation that HSCs do not engraft as they transit S/G2/M. Recently identified candidate inhibitors of human HSC cycling are transforming growth factor-β1(TGF-β1) and stroma-derived factor–1 (SDF-1). To determine the ability of these factors to alter the transplantability of human HSCs proliferating in vitro, lin− cord blood cells were first cultured for 96 hours in serum-free medium containing Flt3 ligand, Steel factor, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. These cells were then transferred to medium containing Steel factor and thrombopoietin with or without SDF-1 and/or TGF-β1 for 48 hours. Exposure to SDF-1 but not TGF-β1 significantly increased (> 2-fold) the recovery of HSCs able to repopulate nonobese diabetic/severe combined immunodeficiency mice. These results suggest new strategies for improving the engraftment activity of HSCs stimulated to proliferate ex vivo.

Recreating a Human Hematopoietic Stem Cell Niche in Vitro by Unique Stroma Cells from the First Trimester Human Placenta and Fetal Liver

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3568-3568
Author(s):  
Mattias Magnusson ◽  
Melissa Romero ◽  
Sacha Prashad ◽  
Ben Van Handel ◽  
Suvi Aivio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Placenta ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Embryonic Stem ◽  
Cell Types ◽  
First Trimester ◽  
Hematopoietic Stem ◽  
Human Hscs

Abstract Expansion of human hematopoietic stem cells (HSCs) ex vivo has been difficult due to limited understanding of their growth requirements and the molecular complexity of their natural microenvironments. To mimic the niches in which human HSCs normally develop and expand during ontogeny, we have derived two unique types of stromal niche cells from the first trimester human placenta and the fetal liver. These lines either support maintenance of multipotential progenitors in culture, or promote differentiation into macrophages. Impressively, the supportive lines facilitate over 50,000-fold expansion of the most immature human HSCs/progenitors (CD34+CD38-Thy1+) during 8-week culture supplemented with minimal cytokines FLT3L, SCF and TPO, whereas the cells cultured on non-supportive stroma or without stroma under the same conditions differentiated within 2 weeks. As the supportive stroma lines also facilitate differentiation of human hematopoietic progenitors into myeloid, erythroid and B-lymphoid lineages, we were able to show that the expanded progenitors preserved full multipotentiality during long-term culture ex vivo. Furthermore, our findings indicate that the supportive stroma lines also direct differentiation of human embryonic stem cells (hESC) into hematopoietic progenitor cells (CD45+CD34+) that generate multiple types of myeloerythroid colonies. These data imply that the unique supportive niche cells can both support hematopoietic specification and sustain a multilineage hematopoietic hierarchy in culture over several weeks. Strikingly, the supportive effect from the unique stromal cells was dominant over the differentiation effect from the non-supportive lines. Even supernatant from the supportive lines was able to partially protect the progenitors that were cultured on the non-supportive lines, whereas mixing of the two types of stroma resulted in sustained preservation of the multipotential progenitors. These results indicate that the supportive stroma cells possess both secreted and surface bound molecules that protect multipotentiality of HSCs. Global gene expression analysis revealed that the supportive stroma lines from both the placenta and the fetal liver were almost identical (r=0.99) and very different from the non-supportive lines that promote differentiation (r=0.34), implying that they represent two distinct niche cell types. Interestingly, the non-supportive lines express known mesenchymal markers such as (CD73, CD44 and CD166), whereas the identity of the supportive cells is less obvious. In summary, we have identified unique human stromal niche cells that may be critical components of the HSC niches in the placenta and the fetal liver. Molecular characterization of these stroma lines may enable us to define key mechanisms that govern the multipotentiality of HSCs.

Hoxb4-YFP reporter mouse model: a novel tool for tracking HSC development and studying the role of Hoxb4 in hematopoiesis

Blood ◽  
2011 ◽  
Vol 117 (13) ◽  
pp. 3521-3528 ◽  
Author(s):  
David Hills ◽  
Ruby Gribi ◽  
Jan Ure ◽  
Natalija Buza-Vidas ◽  
Sidinh Luc ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Hox Genes ◽  
Fetal Liver ◽  
Yellow Fluorescent Protein ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Hematopoietic Stem ◽  
Reporter Mouse

Abstract Hoxb4 overexpression promotes dramatic expansion of bone marrow (BM) hematopoietic stem cells (HSCs) without leukemic transformation and induces development of definitive HSCs from early embryonic yolk sac and differentiating embryonic stem cells. Knockout studies of Hoxb4 showed little effect on hematopoiesis, but interpretation of these results is obscured by the lack of direct evidence that Hoxb4 is expressed in HSCs and possible compensatory effects of other (Hox) genes. To evaluate accurately the pattern of Hoxb4 expression and to gain a better understanding of the physiologic role of Hoxb4 in the hemato-poietic system, we generated a knock-in Hoxb4–yellow fluorescent protein (YFP) reporter mouse model. We show that BM Lin−Sca1+c-Kit+ cells express Hoxb4-YFP and demonstrate functionally in the long-term repopulation assay that definitive HSCs express Hoxb4. Similarly, aorta-gonad-mesonephrous–derived CD45+CD144+ cells, enriched for HSCs, express Hoxb4. Furthermore, yolk sac and placental HSC populations express Hoxb4. Unexpectedly, Hoxb4 expression in the fetal liver HSCs is lower than in the BM, reaching negligible levels in some HSCs, suggesting an insignificant role of Hoxb4 in expansion of fetal liver HSCs. Hoxb4 expression therefore would not appear to correlate with the cycling status of fetal liver HSCs, although highly proliferative HSCs from young BM show strong Hoxb4 expression.

FLT3 receptor and ligand are dispensable for maintenance and posttransplantation expansion of mouse hematopoietic stem cells

Blood ◽  
2009 ◽  
Vol 113 (15) ◽  
pp. 3453-3460 ◽  
Author(s):  
Natalija Buza-Vidas ◽  
Min Cheng ◽  
Sara Duarte ◽  
Hojjatollah Nozad Charoudeh ◽  
Sten Eirik W. Jacobsen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Steady State ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Functional Evaluation ◽  
Hematopoietic Stem ◽  
First Time ◽  
Deficient Mice

Abstract Originally cloned from hematopoietic stem cell (HSC) populations and its ligand being extensively used to promote ex vivo HSC expansion, the FMS-like tyrosine kinase 3 (FLT3; also called FLK2) receptor and its ligand (FL) were expected to emerge as an important physiologic regulator of HSC maintenance and expansion. However, the role of FLT3 receptor and ligand in HSC regulation remains unclear and disputed. Herein, using Fl-deficient mice, we establish for the first time that HSC expansion in fetal liver and after transplantation is FL independent. Because previous findings in Flk2−/− mice were compatible with an important role of FLT3 receptor in HSC regulation and because alternative ligands might potentially interact directly or indirectly with FLT3 receptor, we here also characterized HSCs in Flk2−/− mice. Advanced phenotypic as well as functional evaluation of Flk2−/− HSCs showed that the FLT3 receptor is dispensable for HSC steady-state maintenance and expansion after transplantation. Taken together, these studies show that the FLT3 receptor and ligand are not critical regulators of mouse HSCs, neither in steady state nor during fetal or posttransplantation expansion.

The AC133+CD38−, but not the rhodamine-low, phenotype tracks LTC-IC and SRC function in human cord blood ex vivo expansion cultures

Blood ◽  
2010 ◽  
Vol 115 (2) ◽  
pp. 257-260 ◽  
Author(s):  
Caryn Y. Ito ◽  
Daniel C. Kirouac ◽  
Gerard J. Madlambayan ◽  
Mei Yu ◽  
Ian Rogers ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Ex Vivo ◽  
Hematopoietic Progenitors ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Expansion System ◽  
And Function ◽  
Limiting Dilution

Abstract Phenotypic markers associated with human hematopoietic stem cells (HSCs) were developed and validated using uncultured cells. Because phenotype and function can be dissociated during culture, better markers to prospectively track and isolate HSCs in ex vivo cultures could be instrumental in advancing HSC-based therapies. Using an expansion system previously shown to increase hematopoietic progenitors and SCID-repopulating cells (SRCs), we demonstrated that the rhodamine-low phenotype was lost, whereas AC133 expression was retained throughout culture. Furthermore, the AC133+CD38− subpopulation was significantly enriched in long-term culture-initiating cells (LTC-IC) and SRCs after culture. Preculture and postculture analysis of total nucleated cell and LTC-IC number, and limiting dilution analysis in NOD/SCID mice, showed a 43-fold expansion of the AC133+CD38− subpopulation that corresponded to a 7.3-fold and 4.4-fold expansion of LTC-ICs and SRCs in this subpopulation, respectively. Thus, AC133+CD38− is an improved marker that tracks and enriches for LTC-IC and SRC in ex vivo cultures.

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