Delineation of T-progenitor cell activity within the CD34+ compartment of adult bone marrow

T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T-progenitor cell potential within the CD34+ Lineage-- (Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin-ABM cells with CD45RA, Thy-1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC-containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA-cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro.

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The γ-globin gene promoter progressively demethylates as the hematopoietic stem progenitor cells differentiate along the erythroid lineage in baboon fetal liver and adult bone marrow

Experimental Hematology ◽

10.1016/j.exphem.2006.09.001 ◽

2007 ◽

Vol 35 (1) ◽

pp. 48-55 ◽

Cited By ~ 9

Author(s):

Mahipal Singh ◽

Donald Lavelle ◽

Kestis Vaitkus ◽

Nadim Mahmud ◽

Maria Hankewych ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Fetal Liver ◽

Globin Gene ◽

Gene Promoter ◽

Hematopoietic Stem ◽

Adult Bone Marrow ◽

Adult Bone

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Thrombopoietin Is Synthesized by Bone Marrow Stromal Cells

Blood ◽

10.1182/blood.v90.9.3444 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3444-3455 ◽

Cited By ~ 72

Author(s):

Anastasia Guerriero ◽

Lydia Worford ◽

H. Kent Holland ◽

Gui-Rong Guo ◽

Kevin Sheehan ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Stromal Cells ◽

Cell Sorting ◽

Bone Marrow Cells ◽

Adult Bone Marrow ◽

Hla Dr ◽

Adult Bone ◽

Marrow Cells

Abstract We have previously characterized stromal progenitor cells contained in fetal bone marrow by fluorescence-activated cell sorting (FACS) using the differential expression of CD34, CD38, and HLA-DR, and found that a small number were contained within the CD34+ cell fraction. In the present study, the frequency of stromal progenitors in both the CD34+ and CD34− subpopulations from samples of fetal and adult bone marrow was approximately one in 5,000 of the mononuclear cell fraction. Using multiparameter single-cell sorting, one in 20 fetal bone marrow cells with the CD34+, CD38−, HLA-DR−, CDw90+ phenotype were clonogenic stromal progenitors, whereas greater than one in five single cells with the CD34−, CD38−, HLA-DR−, CDw90+ phenotype formed stromal cultures. We found that cultures initiated by hematopoietic and stromal progenitors contained within the CD34+ fraction of bone marrow cells formed mixed hematopoietic/stromal cell cultures that maintained the viability of the hematopoietic progenitor cells for 3 weeks in the absence of added hematopoietic cytokines. We characterized some of the hematopoietic cytokines synthesized by stromal cultures derived from either CD34+ or CD34− bone marrow cells using reverse transcriptase–polymerase chain reaction (RT-PCR) amplification of interleukin-3 (IL-3), stem cell factor (SCF), CD34, Flt3/Flk2 ligand (FL), and thrombopoietin (TPO) mRNA sequences. We found ubiquitous expression of TPO mRNA in greater than 90% of stromal cultures initiated by either CD34+ or CD34− cells, and variable expression of SCF, FL, and CD34 mRNA. In particular, SCF and CD34 mRNA were detected only in stromal cultures initiated by CD34+ bone marrow cells, although the differences between CD34+ and CD34− stromal cells were not statistically significant. IL-3 mRNA was not found in any stromal cultures. An enzyme-linked immunosorbent assay (ELISA) of soluble SCF and TPO present in culture supernatants demonstrated that biologically significant amounts of protein were secreted by some cultured stromal cells: eight of 16 samples of conditioned media from stromal cultures initiated by fetal and adult bone marrow contained more than 32 pg/mL SCF (in the linear range of the ELISA), with a median value of 32 pg/mL (range, 9 to 230), while 13 of 24 samples of conditioned media had more than 16 pg/mL TPO (in the linear range of the ELISA), with a median of 37 pg/mL (range, 16 to 106). Our data indicate that stromal cultures initiated by single bone marrow cells can make FL, SCF, and TPO. Local production of early-acting cytokines and TPO by stromal cells may be relevant to the regulation of hematopoietic stem cell self-renewal and megakaryocytopoiesis in the bone marrow microenvironment.

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NOTCH1 Induces Differential Epigenomic Patterning and Genomic Organization in Fetal Liver- and Adult Bone Marrow-Derived Hematopoietic Progentiors

Blood ◽

10.1182/blood.v126.23.3637.3637 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3637-3637

Author(s):

Vincenzo Giambra ◽

Sonya H Lam ◽

Miriam Belmonte ◽

Sam Gusscott ◽

Sohrab Salehi ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Promoter Region ◽

Fetal Liver ◽

Lymphoblastic Leukemia ◽

Chromosome Conformation Capture ◽

Chromosome Conformation ◽

Hematopoietic Stem ◽

Adult Bone Marrow ◽

Adult Bone

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T-cell progenitors, characterized by activating NOTCH1 mutations in over 50% of children and adult cases. Although intensive multiagent chemotherapy achieves cure in most pediatric patients, the majority of adults succumb quickly to their disease. The basis for this divergence is likely multifactorial, but we sought in this study to investigate whether cell intrinsic features might contribute to the disparate biologies in pediatric and adult patients. In our prior abstract, we modeled pediatric and adult leukemias by transduction of hematopoietic stem/progenitor cells (HSPC) derived from mouse fetal liver (FL) and adult bone marrow (ABM) with activated NOTCH1 virus followed by transplantation into histocompatible recipient animals. We observed that whereas FL- and ABM-derived HSPC generate similar primary acute T-cell leukemias in terms of penetrance, latency, disease burden/distribution, and immunophenotype, FL leukemias exhibit much greater cycling activity than ABM leukemias, yet are dramatically impaired in their ability to propagate disease in secondary and tertiary recipients compared to ABM leukemias. Using a combination of gene expression profiling and in vitro culture assays, we attributed this differential behavior to NOTCH1-induced autocrine IGF signaling that is operative in FL, but not ABM-derived HSPC. Here we report that NOTCH1 mediates its effects on IGF1 in FL-derived HSPC directly by physical occupancy over the IGF1 promoter in a dimerization-dependent fashion. As well, increased NOTCH1 occupancy at the IGF1 promoter region in FL tissues is associated with reduced histone H3K27 trimethylation (a mark of transcriptionally silent chromatin), yet there is equivalent histone H3K4 trimethylation (a mark identifying transcriptionally active promoters) in both FL and ABM tissues, suggesting that NOTCH1 may be responsible for interconverting the IGF1 locus between active and inactive, but poised chromatin states. NOTCH1 occupancy is also associated with enhanced physical interactions between the IGF1 promoter region and distant genomic loci as revealed by circularized chromosome conformation capture (4C) assay and confirmed by chromosome conformation capture (3C) assay, including sites with H3K4 monomethylation (a mark of transcriptional enhancers) suggesting that NOTCH1 promotes "looping in" of distant enhancer elements that drive IGF1 expression in FL tissues. We conclude from these studies that NOTCH1 enacts differential, developmental stage-specific transcriptional programs by a combination of local epigenetic patterning and long-range genomic interactions. These findings support the notion that pediatric and adult T-ALL may potentially be regarded as related, but biologically distinct diseases, and that novel, age-specific therapies that exploit these differences may improve clinical outcomes. Disclosures No relevant conflicts of interest to declare.

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Generation of T cells from cytokine-mobilized peripheral blood and adult bone marrow CD34+ cells

Blood ◽

10.1182/blood.v84.1.104.104 ◽

1994 ◽

Vol 84 (1) ◽

pp. 104-110 ◽

Cited By ~ 14

Author(s):

AH Galy ◽

S Webb ◽

D Cen ◽

LJ Murray ◽

J Condino ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Cell Activity ◽

Immunodeficient Mice ◽

Adult Bone Marrow ◽

Cd34 Cells ◽

Adult Bone ◽

Mobilized Peripheral Blood

Abstract The present study compared the T-cell progenitor content of CD34+ lineage (Lin)- cells isolated from normal adult bone marrow (ABM) and mobilized peripheral blood (MPB). Both cell populations were found to differentiate into T cells when injected into human fetal thymi implanted into severe combined immunodeficient mice. Cytokine-MPB cells were less efficient than ABM cells in engrafting in the fetal human thymus, although both gave rise to thymocytes with identical phenotypes based on the analysis of CD1a, CD3, CD4, and CD8 expression. Thymocytes derived from adult CD34+ Lin- cells were capable of fully differentiating into mature CD3+ T cells expressing either the T-cell receptor (TCR) gamma delta or the TCR alpha beta (the later associated with CD4 or CD8), showing that the T-cell progenies of adult CD34+ cells were polyclonal and functional. Our data indicate that human MPB CD34+ cells are qualitatively identical to their BM counterparts, and demonstrate the existence of T-lymphoid progenitor cell activity in MPB.

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Generation of T cells from cytokine-mobilized peripheral blood and adult bone marrow CD34+ cells

Blood ◽

10.1182/blood.v84.1.104.bloodjournal841104 ◽

1994 ◽

Vol 84 (1) ◽

pp. 104-110

Author(s):

AH Galy ◽

S Webb ◽

D Cen ◽

LJ Murray ◽

J Condino ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Cell Activity ◽

Immunodeficient Mice ◽

Adult Bone Marrow ◽

Cd34 Cells ◽

Adult Bone ◽

Mobilized Peripheral Blood

The present study compared the T-cell progenitor content of CD34+ lineage (Lin)- cells isolated from normal adult bone marrow (ABM) and mobilized peripheral blood (MPB). Both cell populations were found to differentiate into T cells when injected into human fetal thymi implanted into severe combined immunodeficient mice. Cytokine-MPB cells were less efficient than ABM cells in engrafting in the fetal human thymus, although both gave rise to thymocytes with identical phenotypes based on the analysis of CD1a, CD3, CD4, and CD8 expression. Thymocytes derived from adult CD34+ Lin- cells were capable of fully differentiating into mature CD3+ T cells expressing either the T-cell receptor (TCR) gamma delta or the TCR alpha beta (the later associated with CD4 or CD8), showing that the T-cell progenies of adult CD34+ cells were polyclonal and functional. Our data indicate that human MPB CD34+ cells are qualitatively identical to their BM counterparts, and demonstrate the existence of T-lymphoid progenitor cell activity in MPB.

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Fetal Vs. Adult Hematopoietic Progenitors Respond Differently to NOTCH1: Implications for Pediatric Vs. Adult T-ALL.

Blood ◽

10.1182/blood.v120.21.2381.2381 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2381-2381

Author(s):

Vincenzo Giambra ◽

Sonya H Lam ◽

Amy Ng ◽

Claudia Benz ◽

Olena O Shevchuk ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Lymphoblastic Leukemia ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Adult Bone Marrow ◽

Old Adult ◽

Adult Bone

Abstract Abstract 2381 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of T-cell progenitors which affects both children and adults. Whereas pediatric T-ALL is curable in 80–90% of cases, only 40% of adults with T-ALL survive beyond 5 years. Fetal liver and adult bone marrow-derived hematopoietic stem/progenitor cells (HSPCs) are known to differ in terms of their gene expression programs and functional properties. Despite this work, the extent to which differential programming of fetal and adult HSPCs may impact the biology of their respective leukemias in children and adults remains unexplored. NOTCH1 is a prominent oncogene in T-ALL and activated by mutation in over 50% of cases. The retroviral NOTCH1 mouse bone marrow transplant model of T-ALL is well established; however, most investigators use 8–12 week-old adult mice as bone marrow donors for these experiments and thus these studies could be interpreted as having modeled adult disease. In order to explore the possibility that fetal program HSPCs would more accurately model pediatric disease, we transduced lineage-negative fetal liver HSPCs with activated NOTCH1 (ΔE) retrovirus, transplanted them into syngeneic (C57BL/6) recipients, and compared the behavior of the resulting leukemias to those generated from lineage-negative 8-week-old adult bone marrow HSPCs. Primary transplant recipient mice developed nearly identical T-ALL disease in terms of penetrance, latency, disease distribution/burden, and immunophenotype. Serial transplantation of these leukemias into secondary recipients, however, revealed stark differences in that whereas “adult” leukemias were readily transplantable, “fetal” leukemias were largely non-transplantable. In order to quantitate leukemia-initiating cell (LIC) frequencies in these two situations, we performed secondary transplants into highly permissive, immunodeficient (NOD/Scid/Il2rg−/−) recipients at limiting dilution and observed fetal leukemias to exhibit 500-fold lower LIC activity than adult leukemias (1 in ∼4500 cells vs. 1 in ∼9 cells, respectively). To identify potential mechanisms that might underlie this difference in LIC activity, we compared the behaviors of fetal liver vs. adult bone marrow-derived HSPCs shortly after transduction with NOTCH1(ΔE) virus. Interestingly, NOTCH1 induced fetal HSPCs to cycle rapidly whereas adult HSPCs were largely quiescent. We also noted that non-transduced cells in fetal HSPC cultures were also cycling rapidly, and through a series of fetal/adult mixing and conditioned media experiments, we determined that NOTCH1 induces an autocrine IGF1 signaling circuit in fetal, but not adult HSPCs. This observation was also confirmed to hold true for CD34+ human cord blood vs. adult bone marrow HSPCs. Expression profiling/qRT-PCR and chromatin immunoprecipitation (ChIP) studies further revealed NOTCH1 to induce IGF1 transcription and altered chromatin structure (increased H3K4me3 and decreased H3K27me3 marks) specifically in fetal, but not adult HSPCs. These findings suggest that developmental stage-specific programming in fetal vs. adult progenitors underlies their differential responses to oncogenic NOTCH1 signaling, and also the biological aggressiveness of resulting leukemias. Therapeutic targeting of adult-specific pathways may thus achieve improved clinical responses in adults with T-ALL and perhaps also the minority of pediatric patients with more aggressive, possibly “adult-like” disease. Disclosures: No relevant conflicts of interest to declare.

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Transplanted adult hematopoietic stems cells differentiate into functional endothelial cells

Blood ◽

10.1182/blood-2003-05-1684 ◽

2004 ◽

Vol 103 (1) ◽

pp. 13-19 ◽

Cited By ~ 188

Author(s):

Alexis S. Bailey ◽

Shuguang Jiang ◽

Michael Afentoulis ◽

Christina I. Baumann ◽

David A. Schroeder ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Endothelial Cell ◽

Hematopoietic Stem Cells ◽

Blood Vessels ◽

Cell Potential ◽

Hematopoietic Stem ◽

Adult Bone Marrow ◽

Adult Bone

Abstract During early embryogenesis, blood vessels and hematopoietic cells arise from a common precursor cell, the hemangioblast. Recent studies have identified endothelial progenitor cells in the peripheral blood, and there is accumulating evidence that a subset of these cells is derived from precursors in the bone marrow. Here we show that adult bone marrow–derived, phenotypically defined hematopoietic stem cells (c-kit+, Sca-1+, lineage–) give rise to functional endothelial cells. With the exception of the brain, donor-derived cells are rapidly integrated into blood vessels. Durably engrafted endothelial cells express CD31, produce von Willebrand factor, and take up low-density lipoprotein. Analysis of DNA content indicates that donor-derived endothelial cells are not the products of cell fusion. Self-renewal of stem cells with hematopoietic and endothelial cell potential was revealed by serial transplantation studies. The clonal origin of both hematopoietic and endothelial cell outcomes was established by the transfer of a single cell. These results suggest that adult bone marrow–derived hematopoietic stem cells may serve as a reservoir for endothelial cell progenitors.

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Hematopoietic progenitor cell regulation by CD4+CD25+ T cells

Blood ◽

10.1182/blood-2009-04-218826 ◽

2010 ◽

Vol 115 (23) ◽

pp. 4934-4943 ◽

Cited By ~ 25

Author(s):

Maite Urbieta ◽

Isabel Barao ◽

Monica Jones ◽

Roland Jurecic ◽

Angela Panoskaltsis-Mortari ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Cell Activity ◽

Innate Immune ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem

Abstract CD4+CD25+FoxP3+ regulatory T cells (Tregs) possess the capacity to modulate both adaptive and innate immune responses. We hypothesized that Tregs could regulate hematopoiesis based on cytokine effector molecules they can produce. The studies here demonstrate that Tregs can affect the differentiation of myeloid progenitor cells. In vitro findings demonstrated the ability of Tregs to inhibit the differentiation of interleukin-3 (IL-3)/stem cell factor (colony-forming unit [CFU]-IL3)–driven progenitor cells. Inhibitory effects were mediated by a pathway requiring cell-cell contact, major histocompatibility complex class II expression on marrow cells, and transforming growth factor-β. Importantly, depletion of Tregs in situ resulted in enhanced CFU-IL3 levels after bone marrow transplantation. Cotransplantation of CD4+FoxP3+gfp Tregs together with bone marrow was found to diminish CFU-IL3 responses after transplantation. To address the consequence of transplanted Tregs on differentiated progeny from these CFU 2 weeks after hematopoietic stem cell transplantation, peripheral blood complete blood counts were performed and examined for polymorphonuclear leukocyte content. Recipients of cotransplanted Tregs exhibited diminished neutrophil counts. Together, these findings illustrate that both recipient and donor Tregs can influence hematopoietic progenitor cell activity after transplantation and that these cells can alter responses outside the adaptive and innate immune systems.

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