Functional analysis of the Human Pbx Interacting Protein (HPIP) in Human Hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2430-2430
Author(s):  
Pawandeep Kaur ◽  
Christiane Stadler ◽  
Farid Ahmed ◽  
Monica Cusan ◽  
R. Keith Humphries ◽  
...  
Keyword(s):  
Cord Blood ◽  
Cell Fate ◽  
Constitutive Expression ◽  
Homeodomain Protein ◽  
Acute Lymphocytic Leukaemia ◽  
Human Cord Blood ◽  
Interacting Protein ◽  
Hematopoietic Stem

Abstract The recognition of novel proteins that regulate human hematopoietic stem cell and early progenitor cell fate is a prime objective in experimental and clinical hematology. Human hematopoietic PBX interacting protein (HPIP), with no significant homology to known proteins, is a 731 amino acid protein, discovered as a novel interacting partner of the PBX homeodomain protein. HPIP has been implicated as a nuclear-cytoplasmic shuttle molecule and shown to have the capacity to bind to the cytoskeleton. It also inhibits the ability of PBX-HOX heterodimers to bind to target sequences and strongly inhibits the transactivation activity of E2A-PBX1 [t(1;19) translocation, which occurs in 25% of pediatric pre-B cell acute lymphocytic leukaemia] (Abramovich C. et al JBC, 2000; Oncogene, 2002). It is highly expressed in human CD34+ progenitor cells, but is silenced in differentiated cells. To gain further insights into the possible functional role of HPIP and its domains and its possible role in a common pathway with HOX transcription co-factor PBX1, HPIP cDNA was cloned in pMSCV-IRES-YFP cassette. Umbilical cord blood enriched with CD34+ population of stem cells was obtained to perform in vitro and in vivo experiments. Mutants, with deletions of the microtubule binding region (ΔMBR-HPIP), and nuclear receptor and PBX1 interacting motif (ΔNRPID-HPIP) were generated and tested in vitro and in vivo. The constitutive expression of HPIP wt and ΔMBR-HPIP in human cord blood cells (CD34+) enhanced erythroid colony formation in CFC assay (p=0.008, n=6) while the ΔNRPID-HPIP mutant nullified the effect. Both mutants of HPIP augmented significantly, the formation of primitive colonies (GEMM and GM) in methylcellulose assay (p≤0.01, n=6) as compared to YFP control and HPIP wt. In replating CFC assays ΔNRPID-HPIP showed an increased number of myeloid colonies (p≤0.01, n=6) and GM (p=ns) colonies but a decrease in granulocytic colonies (p≤0.05, n=6) compared to YFP control and HPIP wt

High GATA-2 expression inhibits human hematopoietic stem and progenitor cell function by effects on cell cycle

Blood ◽  
2009 ◽  
Vol 113 (12) ◽  
pp. 2661-2672 ◽  
Author(s):  
Alex J. Tipping ◽  
Cristina Pina ◽  
Anders Castor ◽  
Dengli Hong ◽  
Neil P. Rodrigues ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cord Blood ◽  
Cell Function ◽  
Ki 67 ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cord Blood Cells ◽  
Low Efficiency

Abstract Evidence suggests the transcription factor GATA-2 is a critical regulator of murine hematopoietic stem cells. Here, we explore the relation between GATA-2 and cell proliferation and show that inducing GATA-2 increases quiescence (G0 residency) of murine and human hematopoietic cells. In human cord blood, quiescent fractions (CD34+CD38−HoechstloPyronin Ylo) express more GATA-2 than cycling counterparts. Enforcing GATA-2 expression increased quiescence of cord blood cells, reducing proliferation and performance in long-term culture-initiating cell and colony-forming cell (CFC) assays. Gene expression analysis places GATA-2 upstream of the quiescence regulator MEF, but enforcing MEF expression does not prevent GATA-2–conferred quiescence, suggesting additional regulators are involved. Although known quiescence regulators p21CIP1 and p27KIP1 do not appear to be responsible, enforcing GATA-2 reduced expression of regulators of cell cycle such as CCND3, CDK4, and CDK6. Enforcing GATA-2 inhibited human hematopoiesis in vivo: cells with highest exogenous expression (GATA-2hi) failed to contribute to hematopoiesis in nonobese diabetic–severe combined immunodeficient (NOD-SCID) mice, whereas GATA-2lo cells contributed with delayed kinetics and low efficiency, with reduced expression of Ki-67. Thus, GATA-2 activity inhibits cell cycle in vitro and in vivo, highlighting GATA-2 as a molecular entry point into the transcriptional program regulating quiescence in human hematopoietic stem and progenitor cells.

Identification of a Hierarchy of Multipotent Hematopoietic Progenitors in Human Cord Blood.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2237-2237
Author(s):  
Ravindra Majeti ◽  
Christopher Y. Park ◽  
Irving L. Weissman
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors

Abstract Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC have been identified, as have downstream lineage-committed progenitors, but not multipotent progenitors. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow, and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. While, the function of the CD90- subpopulations is unknown, the CD90+CD45RA- subpopulation presumably contains HSC. We report here in vitro and in vivo functional studies of these three subpopulations from normal human cord blood. In vitro, CD90+CD45RA- cells formed all types of myeloid colonies in methylcellulose and were able to replate with 70% efficiency. CD90-CD45RA- cells also formed all types of myeloid colonies, but replated with only 33% efficiency. CD90-CD45RA+ cells failed to form myeloid colonies in methylcellulose. In liquid culture, CD90+CD45RA- cells gave rise to all three subpopulations; CD90-CD45RA- cells gave rise to both CD90- subpopulations, but not CD90+ cells; CD90-CD45RA+ cells gave rise to themselves only. These data establish an in vitro differentiation hierarchy from CD90+CD45RA- to CD90-CD45RA- to CD90-CD45RA+ cells among Lin-CD34+CD38- cord blood. In vivo, xenotransplantation of CD90+CD45RA- cells into NOD/SCID/IL-2R?-null newborn mice resulted in long-term multilineage engraftment with transplantation of as few as 10 purified cells. Secondary transplants from primary engrafted mice also resulted in long-term multilineage engraftment, indicating the presence of self-renewing HSC. Transplantation of CD90-CD45RA- cells also resulted in long-term multilineage engraftment; however, secondary transplants did not reliably result in long-term engraftment, indicating a reduced capacity for self-renewal. Transplantation of CD90-CD45RA+ cells did not result in any detectable human hematopoietic cells, indicating that the function of these cells is undetermined. Finally, transplantation of limiting numbers of CD90-CD45RA- cells (less than 100) resulted in multilineage human engraftment at 4 weeks, that was no longer detectable by 12 weeks. Thus, the CD90-CD45RA- subpopulation is capable of multilineage differentiation while exhibiting limited self-renewal ability. We believe this study represents the first prospective identification of a population of human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.

scholarly journals Restricted dendritic cell and monocyte progenitors in human cord blood and bone marrow

2015 ◽  
Vol 212 (3) ◽  
pp. 385-399 ◽  
Author(s):  
Jaeyop Lee ◽  
Gaëlle Breton ◽  
Thiago Yukio Kikuchi Oliveira ◽  
Yu Jerry Zhou ◽  
Arafat Aljoufi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cell ◽  
Cord Blood ◽  
Culture System ◽  
Human Monocyte ◽  
Lymphoid Tissues ◽  
Human Cord Blood ◽  
Hematopoietic Stem

In mice, two restricted dendritic cell (DC) progenitors, macrophage/dendritic progenitors (MDPs) and common dendritic progenitors (CDPs), demonstrate increasing commitment to the DC lineage, as they sequentially lose granulocyte and monocyte potential, respectively. Identifying these progenitors has enabled us to understand the role of DCs and monocytes in immunity and tolerance in mice. In humans, however, restricted monocyte and DC progenitors remain unknown. Progress in studying human DC development has been hampered by lack of an in vitro culture system that recapitulates in vivo DC hematopoiesis. Here we report a culture system that supports development of CD34+ hematopoietic stem cell progenitors into the three major human DC subsets, monocytes, granulocytes, and NK and B cells. Using this culture system, we defined the pathway for human DC development and revealed the sequential origin of human DCs from increasingly restricted progenitors: a human granulocyte-monocyte-DC progenitor (hGMDP) that develops into a human monocyte-dendritic progenitor (hMDP), which in turn develops into monocytes, and a human CDP (hCDP) that is restricted to produce the three major DC subsets. The phenotype of the DC progenitors partially overlaps with granulocyte-macrophage progenitors (GMPs). These progenitors reside in human cord blood and bone marrow but not in the blood or lymphoid tissues.

Human reconstituting hematopoietic stem cells up-regulate Fas expression upon active cell cycling but remain resistant to Fas-induced suppression

Blood ◽  
2003 ◽  
Vol 102 (1) ◽  
pp. 118-126 ◽  
Author(s):  
Ingunn Dybedal ◽  
Liping Yang ◽  
David Bryder ◽  
Ingbritt Aastrand-Grundstrom ◽  
Karin Leandersson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Severe Combined Immunodeficiency ◽  
Constitutive Expression ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Nonobese Diabetic

Abstract The Fas receptor and its ligand have been implicated in mediating the bone marrow (BM) suppression observed in graft-versus-host disease and a number of other BM-failure syndromes. However, previous studies have suggested that Fas is probably not expressed on human hematopoietic stem cells (HSCs), but up-regulated as a consequence of their commitment and differentiation, suggesting that progenitors or differentiated blood cells, rather than HSCs, are the targets of Fas-mediated suppression. The present studies confirm that candidate HSCs in human cord blood and BM lack constitutive expression of Fas, but demonstrate that Fas expression on CD34+ progenitor and stem cells is correlated to their cell cycle and activation status. With the use of recently developed in vitro conditions promoting HSC self-renewing divisions, Fas was up-regulated on virtually all HSCs capable of multilineage reconstituting nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice in vivo, as well as on long-term culture-initiating cells (LTC-ICs). Similarly, in vivo cycling of NOD-SCID repopulating cells upon transplantation, resulted in up-regulation of Fas expression. However, repopulating HSCs expressing high levels of Fas remained highly resistant to Fas-mediated suppression, and HSC function was compromised only upon coactivation with tumor necrosis factor. Thus, reconstituting human HSCs up-regulate Fas expression upon active cycling, demonstrating that HSCs could be targets for Fas-mediated BM suppression. (Blood. 2003;102: 118-126)

Enforced expression of MLL-AF4 fusion in cord blood CD34+ cells enhances the hematopoietic repopulating cell function and clonogenic potential but is not sufficient to initiate leukemia

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4746-4758 ◽  
Author(s):  
Rosa Montes ◽  
Verónica Ayllón ◽  
Ivan Gutierrez-Aranda ◽  
Isidro Prat ◽  
M. Carmen Hernández-Lamas ◽  
...  
Keyword(s):  
Cord Blood ◽  
Cell Function ◽  
Lymphoblastic Leukemia ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Dismal Prognosis ◽  
Clonogenic Potential ◽  
The Impact

Abstract Infant acute lymphoblastic leukemia harboring the fusion mixed-lineage leukemia (MLL)-AF4 is associated with a dismal prognosis and very brief latency. Our limited understanding of transformation by MLL-AF4 is reflected in murine models, which do not accurately recapitulate the human disease. Human models for MLL-AF4 disease do not exist. Hematopoietic stem or progenitor cells (HSPCs) represent probable targets for transformation. Here, we explored in vitro and in vivo the impact of the enforced expression of MLL-AF4 in human cord blood-derived CD34+ HSPCs. Intrabone marrow transplantation into NOD/SCID-IL2Rγ−/− mice revealed an enhanced multilineage hematopoietic engraftment, efficiency, and homing to other hematopoietic sites on enforced expression of MLL-AF4. Lentiviral transduction of MLL-AF4 into CD34+ HSPCs increased the in vitro clonogenic potential of CD34+ progenitors and promoted their proliferation. Consequently, cell cycle and apoptosis analyses suggest that MLL-AF4 conveys a selective proliferation coupled to a survival advantage, which correlates with changes in the expression of genes involved in apoptosis, sensing DNA damage and DNA repair. However, MLL-AF4 expression was insufficient to initiate leukemogenesis on its own, indicating that either additional hits (or reciprocal AF4-MLL product) may be required to initiate ALL or that cord blood-derived CD34+ HSPCs are not the appropriate cellular target for MLL-AF4-mediated ALL.

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.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

scholarly journals Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4773-4777 ◽  
Author(s):  
Hal E. Broxmeyer ◽  
Man-Ryul Lee ◽  
Giao Hangoc ◽  
Scott Cooper ◽  
Nutan Prasain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

Unique Differentiation Programs of Human Fetal Liver Stem Cells Shown Both In Vitro and In Vivo in NOD/SCID Mice

Blood ◽  
1999 ◽  
Vol 94 (8) ◽  
pp. 2686-2695 ◽  
Author(s):  
Franck E. Nicolini ◽  
Tessa L. Holyoake ◽  
Johanne D. Cashman ◽  
Pat P.Y. Chu ◽  
Karen Lambie ◽  
...  
Keyword(s):  
Cord Blood ◽  
Fetal Liver ◽  
Scid Mice ◽  
Human Cord Blood ◽  
Human Fetal Liver ◽  
Human Erythropoietin ◽  
Erythroid Precursors ◽  
Lower Ratio

Comparative measurements of different types of hematopoietic progenitors present in human fetal liver, cord blood, and adult marrow showed a large (up to 250-fold), stage-specific, but lineage-unrestricted, amplification of the colony-forming cell (CFC) compartment in the fetal liver, with a higher ratio of all types of CFC to long-term culture-initiating cells (LTC-IC) and a lower ratio of total (mature) cells to CFC. Human fetal liver LTC-IC were also found to produce more CFC in LTC than cord blood or adult marrow LTC-IC, and more of the fetal liver LTC-IC–derived CFC were erythroid. Human fetal liver cells regenerated human multilineage hematopoiesis in NOD/SCID mice with the same kinetics as human cord blood and adult marrow cells, but sustained a high level of terminal erythropoiesis not seen in adult marrow-engrafted mice unless exogenous human erythropoietin (Epo) was injected. This may be due to a demonstrated 10-fold lower activity of murine versus human Epo on human cells, sufficient to distinguish between a differential Epo sensitivity of fetal and adult erythroid precursors. Examination of human LTC-IC, CFC, and erythroblasts generated either in NOD/SCID mice and/or in LTC showed the types of cells and hemoglobins produced also to reflect their ontological origin, regardless of the environment in which the erythroid precursors were generated. We suggest that ontogeny may affect the behavior of cells at many stages of hematopoietic cell differentiation through key changes in shared signaling pathways.

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.

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