Mir-125a Confers Multi-Lineage Long-Term Repopulating Stem Cell Activity to Human Hematopoietic Committed Progenitors

Abstract Recent studies have shown that several miRNA are differentially expressed in hematopoietic stem cells (HSC) and involved in regulating self-renewal, pointing to a new axis of epigenetic control of HSC function. Murine studies have documented a role for miR-125a in regulating HSC as miR-125a enforced expression augments self-renewal. We examined whether these attributes are evolutionarily conserved within human hematopoiesis. Lentiviral vectors over-expressing miR-125a (miR-125OE) were developed and HSC function was investigated using xenotransplantation of CD34+ CD38- human umbilical cord blood (CB) hematopoietic stem and progenitor cells (HSPCs). miR-125OE resulted in significantly increased human bone marrow (BM) chimerism at 12 and 24 weeks post-transplantation and splenomegaly. Within enlarged spleens, there were significantly increased proportions of CD34+CD19+CD10+CD20-B lymphoid cells suggesting a partial B cell differentiation block at the pro-B cell stage. In the BM, CD41+ megakaryocytes, GlyA+ erythroid and CD3+ T cell populations were significantly expanded. Within the primitive compartment, multi-lymphoid progenitors (MLP) were massively expanded by 12 weeks, followed by a combined reduction of immuno-phenotypic HSC and multi-potent progenitors (MPP) by 24 weeks. Given this loss of immuno-phenotypic HSC, we wondered whether stem cell function was compromised in vivo. Secondary transplantation with limiting dilution (LDA) revealed that stem cell frequencies were increased by 4.5 fold in miR-125OE recipients. Using lentivirus sponge-mediated inhibition of miR-125 (miR-125KD) in CD34+CD38-human CB, we were able to directly link these effects to miR-125: B cells increased at the expense of T cells; immuno-phenotypic HSC increased with a concomitant loss of MLP; and functional HSC were decreased by 2.5 fold using secondary LDA assays. Together, these data strongly suggest that miR-125a expression levels regulate human HSC self-renewal and lineage commitment. Since HSC frequency increased so substantially upon miR-125OE, we asked whether more committed cell populations might also be endowed with enhanced self-renewal. Highly purified populations of HSC, MPP and MLP and CD34+CD38+ committed progenitors were transduced and transplanted cells into xenografts. Unexpectedly, miR-125OE transduced CD34+CD38+ progenitors produced a substantial graft after 12 weeks. Control transduced CD34+CD38+ cells did not engraft and only control transduced HSC generated a disseminating graft in recipient mice. miR-125OE transduced HSC and MPP generated robust engraftment, while MLP did not. In all cases, xenografts generated by CD34+CD38+ and MPP transduced with miR-125OE showed multi-lineage repopulation. Moreover, the miR-125OE grafts from CD34+CD38+ and MPP recipients were durable as secondary transplantation generated multi-lineage grafts for at least 20 weeks in 5/7 and 6/10 recipients, respectively; no control transduced groups generated secondary grafts. Thus, the enhancement of self-renewal by enforced expression of miR-125a occurs not only in HSC, but also in MPP and to an as yet unidentified subpopulation within the CD34+38+ committed progenitor compartment. Using protein mass spectrometry, we identified and validated a miR-125a target network in CD34+ CB that normally functions to restrain self-renewal in more committed progenitors. Together, our data suggest that increased miR-125a expression can endow an HSC-like program upon a selected set of non-self-renewing hematopoietic progenitors. Our findings offer the innovative potential to use MPP with enhanced self-renewal to augment limited sources of HSC to improve clinical outcomes. Disclosures No relevant conflicts of interest to declare.

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TIF1γ Knockdown Enhances Hematopoietic Stem Cell Self Renewal with Preferential Myeloid Differentiation and Delayed Erythropoiesis

Blood ◽

10.1182/blood.v118.21.4829.4829 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4829-4829

Author(s):

David C Dorn ◽

Wei He ◽

Joan Massague ◽

Malcolm A.S. Moore

Keyword(s):

Transforming Growth Factor ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Transforming Growth Factor Β ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 4829 The role of TIF1γ in hematopoiesis is still incompletely understood. We previously identified TIF1γ as a novel binding factor for Smad2/3 in the Transforming Growth Factor-β (TFGβ)-inducible signaling pathway implicated in the enhancement of erythropoiesis. To investigate the function of TIF1γ in regulation of hematopoietic stem cells we abrogated TIF1γ signaling by shRNA gamma-retroviral gene transfer in human umbilical cord blood-derived CD34+ hematopoietic stem/ progenitor cells (HCS/ HPCs). Upon blocking TIF1γ the self-renewal capacity of HSCs was enhanced two-fold in vitro as measured by week 5 CAFC assay and three-fold in vivo as measured by competitive engraftment in NOD/ SCID mice over controls. This was associated with a delay in erythroid differentiation and enhanced myelopoiesis. These changes were predominantly observed after TIF1γ knockdown and only mildly after Smad2 depletion but not after Smad3 or 4 reduction. Our data reveal a role for TIF1γ-mediated signaling in the regulation of HSC self-renewal and differentiation. Disclosures: No relevant conflicts of interest to declare.

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Gpx3 Determines Competitiveness of Normal and Leukemic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1587.1587 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1587-1587 ◽

Cited By ~ 1

Author(s):

Olivier Herault ◽

Kristin J Hope ◽

Eric Deneault ◽

Matthias Trost ◽

Nadine Mayotte ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Insertional Mutagenesis ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Cell Activity ◽

Relative Reduction ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 1587 Although important efforts have been invested in the discovery of genes that regulate normal or leukemic hematopoietic stem cells (HSC) self-renewal, the number of validated candidates remains low, due largely to the unavailability of functionally pure stem cell populations. Moreover, it is often difficult to identify the normal counterpart cell from which leukemia originated, further complicating studies based on comparative gene expression. In this study, we used a series of recently characterized Hoxa9 + Meis1 acute myeloid leukemias (AML) derived from fetal liver (FL) cells (Wilhelm BT et al., submitted). These leukemias are remarkably similar in several aspects including their L-HSC frequency (between ∼1 in 100 to 350) except for one leukemia (FLA2) in which 70% of the cells show repopulation ability (i.e., L-HSC). We reasoned that comparative mRNA profiling of FLA2 to the phenotypically similar FLB1 (0.3% L-HSC) might identify genes uniquely associated with L-HSC self-renewal. We observed a 2–3-fold upregulation of Gpx3 in FLA2, which was confirmed by qRT-PCR. In accordance with this, all 14 of the tested Gpx3 promoter region CpG sequences were methylated in FLB1 and hypomethylated in FLA2 cells. The higher expression of GPx3 in FLA2 was confirmed at the protein level and reflected in elevated glutathione peroxidase activity in comparison to FLB1. Importantly, we also observed in FLA2 a relative reduction in reactive oxygen species (ROS) level (DCFDA) and a concomitant decrease in p38 MAPK activation (western blot and mass spectrometry). The correlation of Gpx3 levels with L-HSC frequency could be reflective of their functional dependence on this enzyme. FLA2 cells being difficult to manipulate ex vivo, to address this we utilized retroviruses encoding shRNAs and a GFP reporter to explore the in vivo function of FLA2 cells with downregulated Gpx3. The decrease in percentage of GFP+ donor cells when leukemia became apparent (∼19 days) from that of populations initially transplanted, was 4-fold higher following Gpx3 knockdown in comparison to shLuciferase transduction. Moreover, those shGpx3 infected FLA2 remaining at day 19 displayed a 3-fold decrease in GFP mean fluorescence intensity relative to their control counterparts. These results show that GFPhigh cells were selectively depleted, and suggest that Gpx3 is critical for the competitiveness of L-HSCs. Because redox metabolism has been implicated in HSC self-renewal, we also analyzed its expression and function in normal HSC to gain further insight into the role of GPx3 in stem cell activity. Interestingly, compared to FL-HSCs, isolated 3 and 4 week bone marrow (BM), HSCs exhibited a 39- and 6-fold decrease in Gpx3 expression, respectively. A correlation of Gpx3 levels with enhanced self-renewal was also observed in vitro as overexpression of several nuclear determinants of HSC expansion such as Hoxb4, NA10HD, Klf10 and Prdm16 promoted Gpx3 expression by 3.2 to 19.2-fold. We next infected BM cells enriched for HSCs with retroviruses carrying shRNAs to Gpx3. shRNA targeting of Gpx3 dramatically inhibited hematopoietic reconstitution. Transplantations of sublethally irradiated recipients indicated that Gpx3 knockdown significantly impaired both early and late donor-derived hematopoiesis. These results suggest that GPx3 is critical for repopulation mediated by both short and long-term repopulating cells. In reciprocal gain-of-function experiments, Lin-CD150+CD48- cells engineered to overexpress Gpx3, showed a marked competitive advantage over controls when transplanted following a 7-day ex vivo culture step. Insertional mutagenesis was ruled out as proviral integration analyses of six recipients confirmed polyclonal hematopoiesis. Moreover, some mice were in part reconstituted by the same clones, indicating that self-renewal occurred in vitro prior to transplantation. Phenotypic analysis of late-transplant hematopoietic tissues showed that Gpx3-transduced cells contributed to lymphoid and myeloid repopulation, confirming their multipotentiality. Together, these results indicate that Gpx3 enhances HSC expansion ex vivo possibly through modulation of self-renewal activity. In conclusion, a unique model of primary L-HSC was exploited to identify Gpx3 as a critical determinant for the competitiveness of L-HSCs and complementary experiments demonstrated a key role for this gene in normal HSC self-renewal. Disclosures: No relevant conflicts of interest to declare.

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Vascular Pericytes Sustain Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.2394.2394 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2394-2394 ◽

Cited By ~ 2

Author(s):

Mirko Corselli ◽

Chintan Parekh ◽

Elisa Giovanna Angela Montelatici ◽

Arineh Sahghian ◽

Wenyuan Wang ◽

...

Keyword(s):

Stem Cells ◽

Adipose Tissue ◽

Stem Cell ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Cell Interactions ◽

Lymphoid Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 2394 Mesenchymal stromal (or stem-) cells (MSC) are culture-selected, heterogeneous supporting cells that can differentially regulate hematopoietic stem cell (HSC) behavior in vitro. The elusive identity of native MSC has obscured the contribution, if any, of these cells to HSC support in vivo. Having previously demonstrated that vascular pericytes (ubiquitous cells encircling endothelial cells in capillaries and microvessels) are ancestors of human MSC, we now hypothesize that pericytes are a critical component of the HSC “niche”. Consequently, pericyte isolation from total stroma would allow to develop co-culture systems for human HSC maintenance. In the present study, human cord blood CD34+ cells were cultured onto confluent human pericytes isolated from adipose tissue as CD146+CD34-CD45-CD56- cells. Co-culture of CD34+ cells on pericytes, for up to 6 weeks in the absence of any added growth factor, produced significantly i) higher numbers of CD45+ and CD34+ cells (p<0.05), ii) higher percentages of primitive CD34+CD33-CD10-CD19- progenitors (p<0.05), iii) higher percentages of single- and multi-lineage CFU (p<0.05) and iv) lower percentages of mature myeloid and lymphoid cells (p<0.05), compared to control co-cultures on unfractionated adipose stromal cells (ASC) (n=10 individual experiments, n=4 biological replicates). Most importantly, only pericytes could maintain HSC with self-renewal and long-term repopulating potential, as demonstrated by transplantation into primary and secondary NOD/SCID/IL2Rg−/− mouse recipients (n=3 individual experiments). In the latter setting, none of the mice receiving CD34+ cells co-cultured with ASC engrafted (n=10), whereas all recipients of CD34+ cells cultured in the presence of pericytes developed lympho-myeloid hematopoietic human cells (n=10). Altogether, these results support the hypothesis that pericytes maintain hematopoietic cell stemness. Conversely, unfractionated stromal cell cultures may promote HSC differentiation at the expense of self-renewal. Both tentative scenarios were explored. Co-cultures with pericytes in a transwell system revealed that cell-to-cell contact is required for HSC survival. Since Notch signaling regulates stem cell maintenance by inhibiting cell differentiation through cell-cell interactions, we hypothesized that pericytes purified from total stroma express specific Notch ligands. As shown by qPCR, the expression of Jagged-1 is 2 fold higher in pericytes compared to unfractionated ASC. Addition of a Notch inhibitor (DAPT) to pericyte/HSC co-cultures resulted in the significant reduction of CFU numbers (p<0.05) and increase in B-cell development. Furthermore, increased myeloid differentiation was observed when ASC conditioned medium was added to pericytes/HSC co-cultures. In conclusion, we demonstrate that vascular pericytes sustain HSC by promoting survival and preventing differentiation via cell-to-cell interactions involving Notch activation, whereas unfractionated stroma promotes HSC differentiation through a paracrine mechanism. We thus infer that HSC-supporting stromal cells are not confined within blood-forming organs (similar observations, not reported here, have been made on skeletal muscle pericytes). This novel concept is not easy to reconcile with normal hematopoiesis, but may be highly relevant in the context of the dissemination of malignant hematopoietic cells. Of important note, adipose tissue used in this study represents a convenient, safe and often abundant source of autologous therapeutic cells. Therefore, human fat-derived pericytes emerge as a candidate cell product for HSC ex vivo manipulation in the clinic. Disclosures: No relevant conflicts of interest to declare.

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Hoechst dye efflux reveals a novel CD7+CD34− lymphoid progenitor in human umbilical cord blood

Blood ◽

10.1182/blood.v96.6.2125 ◽

2000 ◽

Vol 96 (6) ◽

pp. 2125-2133 ◽

Cited By ~ 101

Author(s):

Robert W. Storms ◽

Margaret A. Goodell ◽

Alan Fisher ◽

Richard C. Mulligan ◽

Clay Smith

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Large Population ◽

Lymphoid Cells ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract A novel Hoechst 33342 dye efflux assay was recently developed that identifies a population of hematopoietic cells termed side population (SP) cells. In the bone marrow of multiple species, including mice and primates, the SP is composed primarily of CD34−cells, yet has many of the functional properties of hematopoietic stem cells (HSCs). This report characterizes SP cells from human umbilical cord blood (UCB). The SP in unfractionated UCB was enriched for CD34+ cells but also contained a large population of CD34− cells, many of which were mature lymphocytes. SP cells isolated from UCB that had been depleted of lineage-committed cells (Lin− UCB) contained CD34+ and CD34− cells in approximately equivalent proportions. Similar to previous descriptions of human HSCs, the CD34+Lin− SP cells were CD38dimHLA-DRdimThy-1dimCD45RA−CD71−and were enriched for myelo-erythroid precursors. In contrast, the CD34−Lin− SP cells were CD38−HLA-DR−Thy-1−CD71−and failed to generate myelo-erythroid progeny in vitro. The majority of these cells were CD7+CD11b+CD45RA+, as might be expected of early lymphoid cells, but did not express other lymphoid markers. The CD7+CD34−Lin− UCB SP cells did not proliferate in simple suspension cultures but did differentiate into natural killer cells when cultured on stroma with various cytokines. In conclusion, the human Lin− UCB SP contains both CD34+ multipotential stem cells and a novel CD7+CD34−Lin− lymphoid progenitor. This observation adds to the growing body of evidence that CD34− progenitors exist in humans.

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Cellular and molecular characterization of the role of the flk-2/flt-3 receptor tyrosine kinase in hematopoietic stem cells

Blood ◽

10.1182/blood.v84.8.2422.bloodjournal8482422 ◽

1994 ◽

Vol 84 (8) ◽

pp. 2422-2430 ◽

Cited By ~ 8

Author(s):

FC Zeigler ◽

BD Bennett ◽

CT Jordan ◽

SD Spencer ◽

S Baumhueter ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Tyrosine Kinase ◽

Hematopoietic Stem Cell ◽

Receptor Tyrosine Kinase ◽

Lymphoid Cells ◽

Stem Cell Population ◽

Cell Populations ◽

Hematopoietic Stem ◽

Stem Cell Populations

The flk-2/flt-3 receptor tyrosine kinase was cloned from a hematopoietic stem cell population and is considered to play a potential role in the developmental fate of the stem cell. Using antibodies derived against the extracellular domain of the receptor, we show that stem cells from both murine fetal liver and bone marrow can express flk-2/flt-3. However, in both these tissues, there are stem cell populations that do not express the receptor. Cell cycle analysis shows that stem cells that do not express the receptor have a greater percentage of the population in G0 when compared with the flk-2/flt-3- positive population. Development of agonist antibodies to the receptor shows a proliferative role for the receptor in stem cell populations. Stimulation with an agonist antibody gives rise to an expansion of both myeloid and lymphoid cells and this effect is enhanced by the addition of kit ligand. These studies serve to further illustrate the importance of the flk-2/flt-3 receptor in the regulation of the hematopoietic stem cell.

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Akt-Mediated Phosphorylation of Bmi1 Regulates Its Chromatin Association and Growth Promoting Properties.

Blood ◽

10.1182/blood.v114.22.3605.3605 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3605-3605

Author(s):

Yan Liu ◽

Fan Liu ◽

Xinyang Zhao ◽

Goro Sashida ◽

Anthony Deblasio ◽

...

Keyword(s):

Stem Cell ◽

Signaling Pathway ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Akt Signaling ◽

Polycomb Group ◽

Akt Pathway ◽

Akt Signaling Pathway ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 3605 Poster Board III-541 The Polycomb group (PcG) protein Bmi1 maintains silencing of the Ink4a-Arf locus and plays a key role in stem cell self-renewal and oncogenesis. The phosphoinositide 3-kinase-Akt (PI3K-Akt) signaling pathway regulates cell survival, growth, metabolism, migration and angiogenesis. In response to acute Pten loss (which results in Akt activation), mouse embryonic fibroblasts (mefs) accumulate p16Ink4a and p19Arf and undergo senescence. Similarly, Bmi1 −/− mefs undergo premature senescence and accumulate p16Ink4a and p19Arf. PTEN and Bmi1 have similar effects on hematopoiesis; Pten deletion promotes hematopoietic stem cell (HSC) proliferation, resulting in HSC depletion, whereas loss of Bmi1 impairs HSC self-renewal capability, also leading to bone marrow failure. These similarities led us to examine whether the PI3K/Akt pathway functions upstream of Bmi1 to negatively regulate its function and indeed we found that PKB/Akt phosphorylates Bmi1 in vivo, which results in its dissociation from chromatin and in de-repression of the Ink4a-Arf locus. Furthermore, activation of the PI3K/Akt pathway suppresses the ability of Bmi1 to promote cell growth and tumourigenesis and decreases the global level of histone H2A ubiquitination. PI3K/Akt signaling is not active in hematopoietic stem cells, but it is active in more committed progenitor cells. Thus, phosphorylation and inactivation of Bmi1 by Akt may limit HSC self-renewal. Our study also provides a mechanism for the upregulation of p16Ink4a and p19Arf seen in cancer cells that have activation of the PI3K/Akt signaling pathway, and identifies important crosstalk between phosphorylation and chromatin structure. Disclosures: No relevant conflicts of interest to declare.

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MiR-29a Maintains Hematopoietic Stem Cell Self-Renewal and Is Required For Myeloid Leukemogenesis

Blood ◽

10.1182/blood.v122.21.1190.1190 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1190-1190

Author(s):

Wenhuo Hu ◽

James Dooley ◽

Stephen S. Chung ◽

Safak Yalcin ◽

Yu Sup Shin ◽

...

Keyword(s):

Stem Cell ◽

Colony Formation ◽

Target Genes ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Null Mouse ◽

Cell Cycle Regulators ◽

Self Renewal ◽

Hematopoietic Stem

Abstract microRNAs (miRNAs) are important regulators of both embryonic and adult tissue stem cell self-renewal. We previously showed that ectopic expression of miR-29a, a miRNA highly expressed in HSCs as well as in human acute myeloid leukemia (AML) stem cells, in immature mouse hematopoietic cells is sufficient to induce a myeloproliferative disorder that progresses to AML. During the early phase of this disease, miR-29a induces aberrant self-renewal of committed myeloid progenitors, strongly suggesting a role for miR-29a in regulating HSC self-renewal. In order to determine the role of miR-29a in HSC function, we have evaluated our recently described miR-29a/b1 null mouse. Homozygous deletion of miR-29a/b1 resulted in reduced bone marrow cellularity and reduced colony forming capacity of hematopoietic stem and progenitor cells (HSPCs). The phenotype was mediated specifically by miR-29a since miR-29b expression was not significantly altered in HSCs and reconstitution of miR-29a/b1 null HSPCs with miR-29a, but not miR-29b, rescued in vitro colony formation defects. Self-renewal defects were observed in miR-29a deficient HSCs in both competitive and non-competitive transplantation assays, and these deficits were associated with increased HSC cell cycling and apoptosis. Gene expression studies of miR-29a deficient HSCs demonstrated widespread gene dysregulation including a number of up-regulated miR-29a target genes including DNA methylation enzymes (Dnmt3a, -3b) and cell cycle regulators (e.g. Cdk6, Tcl1, Hbp1, Pten). Knockdown of one of these targets, Dnmt3a, in miR-29a deficient HSCs resulted in partial restoration of colony formation, providing functional validation that Dnmt3a mediates part of miR-29a null HSPCs functional defects. miR-29a loss also abrogated leukemogenesis in the MLL-AF9 retroviral AML model. Together, our results demonstrate that miR-29a positively regulates HSC self-renewal and is required for myeloid leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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Identification of a Developmentally-Restricted Hematopoietic Stem Cell That Gives Rise to Innate-like Lymphocytes

Blood ◽

10.1182/blood.v124.21.4302.4302 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4302-4302

Author(s):

Anna E Beaudin ◽

Scott W. Boyer ◽

Gloria Hernandez ◽

Camilla E Forsberg

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Lineage Tracing ◽

Lymphoid Cells ◽

Gfp Expression ◽

Hematopoietic Stem

Abstract The generation of innate-like immune cells distinguishes fetal hematopoiesis from adult hematopoiesis, but the cellular mechanisms underlying differential cell production during development remain to be established. Specifically, whether differential lymphoid output arises as a consequence of discrete hematopoietic stem cell (HSC) populations present during development or whether the fetal/neonatal microenvironment is required for their production remains to be established. We recently established a Flk2/Flt3 lineage tracing mouse model wherein Flk2-driven expression of Cre recombinase results in the irreversible switching of a ubiquitous dual-color reporter from Tomato to GFP expression. Because the switch from Tom to GFP expression in this model involves an irreversible genetic excision of the Tomato gene, a GFP+ cell can never give rise to Tom+ progeny. Using this model, we have definitively demonstrated that all functional, adult HSC remain Tomato+ and therefore that all developmental precursors of adult HSC lack a history of Flk2 expression. In contrast, adoptive transfer experiments of Tom+ and GFP+ fetal liver Lin-cKit+Sca1+ (KLS) fractions demonstrated that both Tom+ and GFP+ fetal HSC support serial, long-term multilineage reconstitution (LTR) in irradiated adult recipients. We have therefore identified a novel, developmentally restricted HSC that supports long-term multilineage reconstitution upon transplantation into an adult recipient but does not normally persist into adulthood. Developmentally-restricted GFP+ HSC display greater lymphoid potential, and regenerated both innate-like B-1 lymphocytes and Vg3-expressing T lymphocytes to a greater extent than coexisting Tom+ FL and adult HSC. Interestingly, whereas developmental regulation of fetal-specific B-cell subsets appears to be regulated cell-instrinsically, as fetal HSC generated more innate-like B-cells than adult HSC even within an adult environment, T-cell development may be regulated both cell intrinsically and extrinsically, as both the cell-of-origin and the fetal microenvironment regulated the generation of innate-like T-cells. Our results provide direct evidence for a developmentally restricted HSC that gives rise to a layered immune system and describes a novel mechanism underlying the source of developmental hematopoietic waves. As early lymphoid cells play essential roles in establishing self-recognition and tolerance, these findings are critical for understanding the development of autoimmune diseases, allergies, and tolerance induction upon organ transplantation. Furthermore, by uncoupling self-renewal capacity in situ with that observed upon transplantation, our data suggests that transplantation- and/or irradiation-induced cues may allow for the engraftment of developmental HSC populations that do not normally persist in situ. As LTR upon transplantation has served as the prevailing definition of adult HSC origin during development, our data challenge the current conceptual framework of adult HSC origin. Disclosures No relevant conflicts of interest to declare.

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Functional properties of cells obtained from human cord blood CD34+ stem cells and mouse cardiac myocytes in coculture

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01285.2007 ◽

2008 ◽

Vol 294 (4) ◽

pp. H1541-H1549 ◽

Cited By ~ 11

Author(s):

Alessia Orlandi ◽

Francesca Pagani ◽

Daniele Avitabile ◽

Giuseppina Bonanno ◽

Giovanni Scambia ◽

...

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Functional Properties ◽

Fluorescent Protein ◽

Cell Populations ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Human Specific

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34+ cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34+ cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days, mononucleated EGFP+ cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca2+] ([Ca2+]i) homeostasis by [Ca2+]i imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP+ cells were connected to surrounding cells by gap junctions, acquired electrophysiological properties similar to those of cardiomyocytes, and showed action potential-associated [Ca2+]i transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca2+]i oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB CD34+ cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, the expression of human-specific cardiac genes was undetectable by RT-PCR.

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Pbx1 Regulates the Self-Renewal of Long-Term Hematopoietic Stem Cells by Maintaining Their Quiescence.

Blood ◽

10.1182/blood.v110.11.94.94 ◽

2007 ◽

Vol 110 (11) ◽

pp. 94-94 ◽

Cited By ~ 1

Author(s):

Francesca Ficara ◽

Mark J. Murphy ◽

Min Lin ◽

Michael L. Cleary

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Acute Leukemia ◽

B Cell ◽

Quiescent State ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cell Fate Decisions

Abstract Pbx1 is a proto-oncogene that was originally discovered at the site of chromosomal translocations in pediatric acute leukemia. It codes for a homeodomain transcription factor, which is a component of hetero-oligomeric protein complexes that regulate developmental gene expression. Lack of Pbx1 is associated with multiple patterning malformations, defects in organogenesis, and severe fetal anemia, however embryonic lethality has prevented an assessment of its roles in the adult hematopoietic stem cell (HSC) compartment and in lymphoid differentiation. The objective of this study was to characterize the physiological roles for Pbx1 in the hematopoietic system, specifically in the regulation of cell fate decisions involved in the timing and/or extent of postnatal HSC and progenitor proliferation, self-renewal or differentiation capacity. A genetic approach was employed to conditionally inactivate Pbx1 in the hematopoietic compartment in vivo using Cre recombinase expressed under the control of the Tie2 or Mx1 promoters. A crucial role for Pbx1 in the development of the lympho-hematopoietic system was evidenced by reduced size, cell number, and altered architectures of the thymus and spleen in mutant mice. A marked reduction was observed in the bone marrow (BM) pro- and pre-B cell compartment, as well as a striking reduction (up to 10-fold) in common lymphoid progenitors (CLP), suggesting a role for Pbx1 at a critical stage of lymphoid development where acute leukemia likely originates. Accordingly, abnormal T cell development was observed in the thymus. Common myeloid progenitors (CMP) and Lin-cKit+Sca1+ (LKS, enriched in HSCs) cells were also reduced, as well as long-term stem cells (LT-HSCs, reduced 7-fold on average). Assessment of the proliferation status of LT- and ST (short-term)-HSCs, as well as multi-potent progenitors (MPP), revealed that the reduction of the HSC compartment was associated with a higher number of stem cells exiting the G0 phase, thus losing their quiescent state. Strikingly, Pbx1-deficient BM cells failed to engraft in competitive transplants, but were able to reconstitute congenic recipients in the absence of competition, indicating a profound defect of functional HSCs, which nevertheless retained reconstitution potential. Importantly, Pbx1 deficient HSCs progressively disappeared from primary transplant recipients, and were unable to engraft secondary recipients, demonstrating that Pbx1 is crucial for the maintenance of LT-HSC self-renewal. Microarray studies performed on mutant and wt LT- and ST-HSCs, followed by bioinformatics analysis, showed that in the absence of Pbx1 LT-HSCs are characterized by premature expression of a large subset of ST-HSC genes. The up-regulated differentially expressed transcripts are enriched for cell cycle regulatory genes, consistent with the observed increased cycling activity. Notably, more than 8% of the down-regulated genes are related to the Tgf-beta pathway, which serves a major role in maintaining HSC quiescence. Moreover, B-cell specific genes, which are expressed in the wt LT-HSC compartment, are down-regulated in the absence of Pbx1, suggesting that the observed reduction in CLP and B-cell numbers ultimately arose from a stem cell defect in lymphoid priming. We conclude that Pbx1 is at the apex of a transcriptional cascade that controls LT-HSC quiescence and differentiation, thus allowing the maintenance of their self-renewal potential, crucial for the homeostasis of the lympho-hematopoietic system.

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