Adult Hematopoietic Progenitor Cells Research Articles

Developing the right tools for the job: Lin28 regulation of early life T-cell development and function.

T cells comprise a functionally heterogeneous cell population that has important roles in the immune system. While T cells are broadly considered to be a component of the antigen-specific adaptive immune response, certain T-cell subsets display innate-like effector characteristics whereas others perform immunosuppressive functions. These functionally diverse T-cell populations preferentially arise at different stages of ontogeny and are tailored to the immunological priorities of the organism over time. Many differences in early life versus adult T-cell phenotypes can be attributed to the cell-intrinsic properties of the distinct progenitors that seed the thymus throughout development. It is becoming clear that Lin28, an evolutionarily conserved, heterochronic RNA-binding protein that is differentially expressed among early life and adult hematopoietic progenitor cells, plays a substantial role in influencing early T-cell development and function. Here, we discuss the mechanisms by which Lin28 shapes the T-cell landscape to protect the developing fetus and newborn. Manipulation of the Lin28 gene regulatory network is being considered as one means of improving hematopoietic stem cell transplant outcomes; as such, understanding the impact of Lin28 on T-cell function is of clinical relevance.

3088 – DISSECTING THE ONTOGENIC REMODELING OF THE PROTEOMIC LANDSCAPE AND FUNCTIONALITY OF HEMATOPOIETIC PROGENITOR CELLS IN NORMAL DEVELOPMENT AND LEUKEMIA

The process of hematopoiesis is subject to extensive ontogenic remodeling that is accompanied by alterations in cellular fate both during normal development and disease. Although the functional differences between fetal and adult hematopoiesis are well established, the responsible molecular mechanisms have long remained largely unexplored on the proteomic level. In our latest work, we characterized and compared the proteomic makeup of fetal and adult HSPCs (Jassinskaja et al., 2017, Cell Reports). Here, we have utilized state-of-the-art mass spectrometry to gain deep coverage of the proteome of 100,000 fetal and adult lymphoid-primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and granulocyte-monocyte progenitors (GMPs). Our analysis resulted in the identification and quantification of over 4000 proteins, with 200-300 proteins per cell type displaying differential expression between the fetus and the adult. We uncovered ontogenic changes in expression of several transcription factors with critical roles in hematopoiesis. Together with other proteins involved in lineage commitment and differentiation, we show that protein expression in fetal and adult hematopoietic progenitor cells is predictive of differential lineage potential. Our subsequent functional analyses further emphasize that the proteomic differences affect cell fate both in the context of normal differentiation and leukemic hematopoiesis driven by the MLL-ENL fusion oncogene. Collectively, our work represents a significant advancement in the understanding of the molecular programs that govern ontogenic differences in hematopoiesis and provides a solid foundation for future investigation of which factors are responsible for the difference in susceptibility and outcome of different leukemias in infants and in adults.

FLT3ITD Target Enhancers Are Primed but Inaccessible in Fetal Hematopoietic Progenitors

The FLT3 Internal Tandem Duplication (FLT3ITD) is common somatic mutation in acute myeloid leukemia (AML). We have previously shown that FLT3ITD fails to induce changes in HSC self-renewal, myelopoiesis and leukemogenesis during fetal stages of life. FLT3ITD signal transduction pathways are hyperactivated in fetal progenitors, but FLT3ITD target genes are not. This suggests that postnatal-specific transcription factors may be required to help induce FLT3ITD target gene expression. Alternatively, repressive histone modifications may impose a barrier to FLT3ITD target gene activation in fetal HPCs that is relaxed during postnatal development. To resolve these possibilities, we used ATAC-seq, as well as H3K4me1, H3K27ac and H3K27me3 ChIP-seq, to identify cis-elements that putatively control FLT3ITD target gene expression in fetal and adult hematopoietic progenitor cells (HPCs). We identified many enhancer elements (ATAC-seq peaks with H3K4me1 and H3K27ac) that exhibited increased chromatin accessibility and activity in FLT3ITD adult HPCs relative to wild type adult HPCs. These elements were enriched near FLT3ITD target genes. HOMER analysis showed enrichment for STAT5, ETS, RUNX1 and IRF binding motifs within the FLT3ITD target enhancers, but motifs for temporally dynamic transcription factors were not identified. We cloned a subset of the enhancers and confirmed that they could synergize with their promoter to activate a luciferase reporter. For representative enhancers, STAT5 binding sites were required to activate the enhancer - as anticipated - and RUNX1 repressed enhancer activity. We tested whether accessibility or priming changed between fetal and adult stages of HPC development. FLT3ITD-dependent changes in chromatin accessibility were not observed in fetal HPCs, though the enhancers were primed early in development as evidenced by the presence of H3K4me1. Repressive H3K27me3 were not present at FLT3ITD target enhancers in either or adult HPCs. The data show that FLT3ITD target enhancers are demarcated early in hematopoietic development, long before they become responsive to FLT3ITD signaling. Repressive marks do not appear to create an epigenetic barrier to enhancer activation in the fetal stage. Instead, age-specific transcription factors are likely required to pioneer enhancer elements so that they can respond to STAT5 and other FLT3ITD effectors. Disclosures No relevant conflicts of interest to declare.

In-Depth Proteomic Characterization of Lineage-Biased Hematopoietic Progenitor Cells in the Fetus and Adult

The process of hematopoiesis is subject to extensive ontogenic remodeling that is accompanied by alterations in cellular fate both during normal development and upon malignant transformation. Although the functional differences between fetal and adult hematopoiesis are well established, the responsible molecular mechanisms have long remained largely unexplored at the proteomic level. We hypothesize that an intrinsically programmed proteomic switch in hematopoietic stem and progenitor cells (HSPCs) during ontogeny regulates the outcome of hematopoiesis both during normal development and upon leukemia initiation, and that the proteomic makeup of the leukemia-initiating cell has an instructive role in determining the outcome of the resulting cancer. In our latest work, we utilized quantitative mass spectrometry-based proteomics to characterize and compare the proteomic makeup of fetal and adult Lin- Sca-1+ cKit+ (LSK) HSPCs (Jassinskaja et al., 2017, Cell Reports), representing all of the earliest stem and progenitors in fetal and adult hematopoiesis. We identified differences in several important cellular processes not previously described to play a role in hematopoiesis, highlighting the need for applying proteomic-centric approaches in the field. In order to further increase our understanding of normal and malignant hematopoiesis during ontogeny, we are now continuing this work by focusing on more stringently defined populations of lineage-biased hematopoietic progenitor cells (HPCs). Here, we have utilized encapsulated methods for preparation of microscale samples in combination with state-of-the-art mass spectrometry to gain deep coverage of the proteome of 100,000 fetal (E14.5) and adult lymphoid-primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and granulocyte-macrophage progenitors (GMPs). Our analysis resulted in the identification and quantification of 4189 proteins, with over 200 proteins per cell type displaying differential expression between the fetus and the adult. Importantly, the differentially expressed proteins were enriched for a broad variety of biological processes. Similar to our previous findings in HSPCs, for all three cell types, proteins higher expressed in the fetus showed a strong enrichment for cell cycle- and translation-related processes, whereas those higher expressed in the adult were enriched for processes related to immune response and redox homeostasis. Our preliminary analysis of hematopoietic cell subset signatures associated with the differentially expressed proteins suggests a stronger lymphoid bias in fetal compared to adult LMPPs as well as CLPs. Surprisingly, the proteomic signature of fetal GMPs suggests a retained megakaryocyte-erythroid potential, which is corroborated by a significantly higher expression of megakaryocyte progenitor marker CD41 on the fetal cells. Upon analyzing expression of transcription factors (TFs) in fetal and adult HPCs, we could confirm differential expression of TFs known to have ontogeny-specific roles in hematopoiesis (e.g. Arid3a and Etv6). Importantly, we also identified several differentially expressed TFs that could represent novel regulators of fetal- and adult-specific features of hematopoiesis, such as Irf8, Btf3, Mndal and Pura. Furthermore, the difference in expression of Irf8 observed here could indicate a previously unknown ontogenic switch in the balance between neutrophil and monocyte production from myeloid-competent progenitors. Lastly, our data shows strong indications of a differential sensitivity towards Rho kinase inhibition between the fetal and the adult HPCs. Collectively, our work represents a significant advancement in the understanding of the molecular programs that govern ontogenic differences in hematopoiesis and provides a solid foundation for future investigation of which factors are responsible for the difference in susceptibility and outcome of different leukemias in infants and in adults. Disclosures No relevant conflicts of interest to declare.

Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies

AbstractWe present a programmable bioengineered 3-dimensional silk-based bone marrow niche tissue system that successfully mimics the physiology of human bone marrow environment allowing us to manufacture functional human platelets ex vivo. Using stem/progenitor cells, megakaryocyte function and platelet generation were recorded in response to variations in extracellular matrix components, surface topography, stiffness, coculture with endothelial cells, and shear forces. Millions of human platelets were produced and showed to be functional based on multiple activation tests. Using adult hematopoietic progenitor cells our system demonstrated the ability to reproduce key steps of thrombopoiesis, including alterations observed in diseased states. A critical feature of the system is the use of natural silk protein biomaterial allowing us to leverage its biocompatibility, nonthrombogenic features, programmable mechanical properties, and surface binding of cytokines, extracellular matrix components, and endothelial-derived proteins. This in turn offers new opportunities for the study of blood component production ex vivo and provides a superior tissue system for the study of pathologic mechanisms of human platelet production.

Phenotypic heterogenecity of hematopoietic progenitor cells from placental tissue: comarative analysis with umbilical cord blood and fetal liver

The study of placental hematopoietic progenitor cells (HPCs) and comparison of their properties with other fetal and adult HPCs is necessary for assessing of their possible clinical application. It has been shown that HPCs from placenta are heterogeneous by phenotype: placental tissue contains three populations with different level of CD34 expression such as CD34+++CD45low/-, CD34++CD45low/- and CD34+/lowCD45low/-. Similar to fetal liver placenta contains both, population of CD34++CD45low/- and CD34+CD45low/-cells, suggesting hematopoiesis in placental tissue. CD34++CD45low/- population also expressed CD133, almost negative for lineage markers, and had lymphocyte-like morphology conforming the presence of primitive HPCs in this population. Additionally, we found later progenitors with phenotype CD34+/lowCD45+ in placental tissueas the majority of these cells expressed hematopoietic lineage markers. Population with phenotype CD34+++CD45low was observed in the placenta that may evidence for their generation in the placental tissue or migration from the other sites of hematopoiesis and changing phenotype under placental microenvironment.

Endothelialization and altered hematopoiesis by persistent Etv2 expression in mice

Etv2 is a master gene for the commitment of hematopoietic/endothelial cells and is a potent inducer of endothelial/hematopoietic cells from embryonic stem cells. Etv2 is highly expressed in endothelial/hematopoietic precursors but is downregulated when they are differentiated, indicating that Etv2 should have transient but not constitutive function. However, relatively little attention has been paid to the importance of transient Etv2 expression. To determine whether transient Etv2 expression is essential to normal development and cell differentiation, we generated mice that constitutively express Etv2 from a Cre-activatable ROSA26 locus in endothelial/hematopoietic, somite, or neuronal lineages. Constitutive Etv2 expression caused profound phenotypes in hematopoietic/endothelial cells, with little effect on somite or neuronal lineages. In hematopoietic/endothelial lineages, constitutive Etv2 expression induced by Tie-2 Cre transgene caused abnormal yolk sac vasculature. Prolonged vascular endothelial cadherin expression and decreased B lymphopoiesis were observed in Etv2 expressing vascular endothelial cadherin(+)/CD45(+) cells, indicating that Etv2 forces endothelial program on hematopoietic cells. Etv2 expression in adult hematopoietic cells by Vav-iCre transgene also conferred an endothelial phenotype on hematopoietic stem cells and suppressed hematopoiesis, with erythropoiesis severely affected. We conclude that constitutive Etv2 expression perturbs vascular development and hematopoiesis. While promoting hematopoiesis/vasculogenesis, Etv2 expression should be tightly regulated to achieve normal vascular development and hematopoiesis.

R-Ras and Rac GTPase Cross-talk Regulates Hematopoietic Progenitor Cell Migration, Homing, and Mobilization

Adult hematopoietic progenitor cells (HPCs) are maintained by highly coordinated signals in the bone marrow. The molecular mechanisms linking intracellular signaling network of HPCs with their microenvironment remain poorly defined. The Rho family GTPase Rac1/Rac2 has previously been implicated in cell functions involved in HPC maintenance, including adhesion, migration, homing, and mobilization. In the present studies we have identified R-Ras, a member of the Ras family, as a key signal mediator required for Rac1/Rac2 activation. We found that whereas Rac1 activity is up-regulated upon stem cell factor, integrin, or CXCL12 stimulation, R-Ras activity is inversely up-regulated. Expression of a constitutively active R-Ras mutant resulted in down-regulation of Rac1-activity whereas deletion of R-Ras led to an increase in Rac1/Rac2 activity and signaling. R-Ras(-/-) HPCs displayed a constitutively assembled cortical actin structure and showed increased directional migration. Rac1/Rac2 inhibition reversed the migration phenotype of R-Ras(-/-) HPCs, similar to that by expressing an R-Ras active mutant. Furthermore, R-Ras(-/-) mice showed enhanced responsiveness to G-CSF for HPC mobilization and exhibited decreased bone marrow homing. Transplantation experiments indicate that the R-Ras deficiency-induced HPC mobilization is a HPC intrinsic property. These results indicate that R-Ras is a critical regulator of Rac signaling required for HPC migration, homing, and mobilization.

An Essential Role of the RNA Editing Enzyme ADAR1 in T Cell Development.

Abstract 917Post-transcriptional regulation such as RNA editing in hematopoiesis and lymphopoiesis is poorly understood. ADAR1 (adenosine deaminase acting on RNA-1) is a RNA editing enzyme essential for embryonic development. Disruption of the ADAR1 gene was shown to cause defective embryonic hematopoiesis (Wang Q et al, Science 2000). Moreover, we have recently obtained direct evidence for the preferential effect of ADAR1 deletion on adult hematopoietic progenitor cells as opposed to the more primitive cells via a RNA-editing dependent mechanism by different conditional gene deletion strategies (Xufeng R et al PNAS 2009, in press). To further determine the role of ADAR1 in T cell development, we generated a mouse model in which ADAR1 was deleted specifically in T lymphocytes by interbreeding ADAR1lox/lox mice with Lck-Cre transgenic mice. In our current study, we report that ADAR1 is essential for T cell differentiation at the late progenitor stage in the thymus, coincident with T cell receptor-α/β expression. In ADAR1lox/loxLck-Cre mice, mature T cells decreased dramatically in peripheral blood, spleen and lymph nodes in comparison to littermate controls. In the thymus, the production of CD4+/CD8+ double positive cells was severely impaired and massive cell death was observed in pre-T cell populations. Within the pro-T cells, ADAR1 deletion resulted in a significant decrease of late progenitor cells but not early progenitor subsets. In both pro-T and pre-T cell stages, defective T cell development preferentially occurred in the beta chain positive cells, but was not apparent in gamma/delta T cells. Our data demonstrated an indispensable role of ADAR1 in early T cell differentiation that correlated with T cell receptor beta chain expression, thereby indicating that RNA editing by ADAR1 is an essential event in T cell development. Disclosures:No relevant conflicts of interest to declare.

ADAR1 is required for hematopoietic progenitor cell survival via RNA editing

Adenosine Deaminase Acting on RNA 1 (ADAR1) is an RNA-editing enzyme that converts adenosine to inosine, following RNA transcription. ADAR1's essential role in embryonic development, especially within the hematopoietic lineage, has been demonstrated in knock-out mice. However, a specific role for ADAR1 in adult hematopoietic progenitor cells (HPCs) remains elusive. In this report, we show that ADAR1 is required for survival of differentiating HPCs as opposed to more primitive cells in adult mice by multiple strategies targeting floxed ADAR1 for deletion by Cre recombinase. As a consequence, ADAR1-deficient hematopoietic stem cells (HSCs) were incapable of reconstituting irradiated recipients although being phenotypically present in the recipient bone marrow. While an effect on HSCs cannot be completely ruled out, the preferential effect of ADAR1 absence on HPCs over more primitive hematopoietic cells was consistent with the increased expression of ADAR1 within HPCs, as well as the inability of ADAR1-deficient HPCs to form differentiated colonies and increased apoptotic fraction during ex vivo culture. Moreover, we have obtained direct evidence that ADAR1 functions in HPCs via an RNA-editing dependent mechanism. Therefore, ADAR1 plays an essential role in adult hematopoiesis through its RNA editing activity in HPCs.

Increased TSLP availability restores T- and B-cell compartments in adult IL-7–deficient mice

AbstractInterleukin 7 (IL-7) plays a crucial role in adult lymphopoiesis, while in fetal life its effect can be partially compensated by TSLP. Whether adult hematopoietic progenitor cells are unresponsive to TSLP or whether TSLP is less available in adult microenvironments is still a matter of debate. Here, we show that increased TSLP availability through transgene (Tg) expression fully restored lymphopoiesis in IL-7–deficient mice: it rescued B-cell development, increased thymic and splenic cellularities, and restored double-negative (DN) thymocytes, αβ and γδ T-cell generation, and all peripheral lymphoid compartments. Analysis of bone marrow chimeras demonstrated that hematopoietic progenitor cells from adult wild-type mice efficiently differentiated toward B- and T-cell lineages in lethally irradiated IL-7 deficient mice provided TSLP Tg was expressed in these mice. In vitro, TSLP promoted the differentiation of uncommitted adult bone marrow progenitors toward B and T lineages and the further differentiation of DN1 and DN2 thymocytes. Altogether, our results show that adult hematopoietic cells are TSLP responsive and that TSLP can sustain long-term adult lymphopoiesis.

Expression of Cadherins in Bone Marrow Stromal Cells Affects Capacity Supporting Expansion of CD34+ Cord Blood Cells.

Ex vivo expansion of hematopoietic progenitor cells (HPCs) has been used to circumvent the limitation of umbilical cord blood applied on adult HPC transplantation and may facilitate ex vivo gene manipulation. Nonetheless, the nature of these signals provided by the stromal microenvironment and mechanisms by which stromal cells regulate the growth of stem cells remain to be defined. With the help of retrovirally-expressing human telomerase reverse transcriptase (hTERT) in marrow stromal cells, we isolated 16 human bone marrow stromal cell lines. After a 7-day short stromal cells-dependent co-culture, the cord blood CD34+ cells were 1.0- to 14.5-fold expanded. Among the adhesion molecules in stromal cells examined, expression of N-cadherin and P-cadherin in stromal cells were found to correlate with capacities of stromal cells to support cord blood HPC expansion and be predictive of HPC-expanding capacities.

PTX-sensitive signals in bone marrow homing of fetal and adult hematopoietic progenitor cells

AbstractSeveral examples suggest a relationship between in vitro migratory capacity and bone marrow (BM) homing. Pertussis toxin (PTX) is a potent inhibitor of serpentine receptor–associated inhibitory trimeric guanidine nucleotide binding (Gi) protein signals. As such, it blocks hematopoietic progenitor cell migration in vitro, but contrary to expectation, no effects on BM homing were observed in previous studies. We therefore re-examined the effect of PTX on homing of murine BM and fetal liver (FL). We found that BM homing of PTX-incubated progenitor cells (colony-forming cells in culture [CFU-Cs]) from BM or FL in irradiated and nonirradiated recipients was reduced by more than 75%, with a concomitant increase in circulating CFU-Cs in peripheral blood. Additional studies confirmed the functional significance of this reduction in homing: PTX-treated cells did not provide radioprotection, and their short-term engraftment in BM and spleen was drastically reduced. Furthermore, several approaches show that cell-intrinsic rather than host-derived mechanisms are responsible for the PTX-induced homing defect. In summary, we show that Gi protein signals are required for BM homing and, as such, provide a new example of the association between BM homing and in vitro migration. Moreover, our data suggest that the behavior of hematopoietic progenitors in obeying Gi signaling does not diverge from that of mature leukocytes.

Total body irradiation causes profound changes in endothelial traffic molecules for hematopoietic progenitor cell recruitment to bone marrow

Nonirradiated bone marrow (BM) venules and sinusoids in murine skull support hematopoietic progenitor cell (HPC) rolling through constitutively expressed endothelial (P- and E-) selectins and VCAM-1. Using intravital microscopy, we tested whether host conditioning with total body irradiation (TBI) changes the molecular mechanisms by which murine HPCs from fetal livers (FL) interact with BM endothelial cells. Although a high dose of TBI did not affect the overall frequency of HPC rolling in BM microvessels, the underlying molecular mechanisms differed from those in nonirradiated BM. TBI induced VCAM-1 up-regulation in BM microvessels, whereas P-selectin expression was reduced and the low baseline level of E-selectin remained unchanged. Only the administration of anti-VCAM-1, but not anti-P- or -E-selectin monoclonal antibodies, decreased FL HPC rolling. Rolling was frequently followed by firm arrest (sticking), even in nonirradiated BM microvessels in which sticking was entirely pertussis toxin-insensitive-that is, Galpha(i)-coupled signaling events (eg, through chemokines) were apparently not required. TBI increased the frequency of sticking FL HPC. This irradiation-induced additional sticking was reversed when FL HPCs were pretreated with pertussis toxin, suggesting that TBI induced elevated expression of a Galpha(i)-protein-coupled chemotactic signal in the BM. This chemoattractant was probably distinct from SDF-1alpha because, unlike adult HPCs, FL HPCs (day 11 of gestation) responded poorly to SDF-1alpha in vitro. These results demonstrate that TBI induces profound changes in the expression of endothelial traffic molecules in the BM, and they indicate that FL HPCs can home to the BM in the absence of SDF-1alpha and other Galpha(i)-protein-coupled signals.

From hematopoietic stem cells to neural stem cells

Stem cells are self-renewable, pluripotent cells that proliferate in adult life by characteristic asymmetric divisions, in which one daughter cell is committed to differentiation whereas the other remains as a stem cell, These cells are also able to differentiate into various cell types under heterotopic environmental influences. In the present study, we have explored the potential of adult hematopoietic bone marrow cells to differentiate into cells of oligodendroglial lineage under physiological, active myelinating conditions. We present evidence of oligodendrocyte generation from adult hematopoietic progenitor cells (CD117+) in vivo, after intracerebral transplantation into the neonatal mouse brain.

Dual Action of Retinoic Acid on Human Embryonic/Fetal Hematopoiesis: Blockade of Primitive Progenitor Proliferation and Shift From Multipotent/Erythroid/Monocytic to Granulocytic Differentiation Program

In preliminary studies, we have analyzed the hematopoietic growth factor (HGF) requirement of hematopoietic progenitor cells (HPCs) purified from embryonic-fetal liver (FL) and grown in fetal calf serum-supplemented (FCS+) clonogenic culture. The key role of erythropoietin (Epo) for colony formation by early erythroid progenitors (burst-forming units-erythroid [BFU-E]) has been confirmed. Furthermore, in the absence of exogenous HGFs, FL monocytic progenitors (colony-forming unit monocyte [CFU-M]) generate large colonies exclusively composed of monocytes-macrophages; these colonies are absent in FCS- clonogenic culture. On this basis, we have investigated the role of all-trans retinoic acid (ATRA) and its isomer 9-cis RA in FL hematopoiesis. Both compounds modulate the growth of purified FL HPCs, which show a dose-dependent shift from mixed/erythroid/ monocytic to granulocytic colony formation. Studies on unicellular and paired daughter cell culture unequivocally indicate that the shift is mediated by modulation of the HPC differentiation program to the granulopoietic pathway (rather than RA-induced down-modulation of multipotent/ erythroid/monocytic HPC growth coupled with recruitment of granulocytic HPCs). ATRA and 9-cis RA also exert their effect on the proliferation of primitive HPCs (high-proliferative potential colony-forming cells [HPP-CFCs]) and putative hematopoietic stem cells (HSCs; assayed in Dexter-type long-term culture). High concentrations of either compound (1) drastically reduced the number of primary HPP-CFC colonies and totally abolished their recloning capacity and (2) inhibited HSC proliferation. It is crucial that these results mirror recent observations indicating that murine adult HPCs transduced with dominant negative ATRA receptor (RAR) gene are immortalized and show a selective blockade of granulocytic differentiation. Altogether, these results suggest that ATRA/9-cis RA may play a key role in FL hematopoiesis via a dual effect hypothetically mediated by interaction with the RAR/RXR heterodimer, ie, inhibition of HSC/ primitive HPC proliferation and induction of CFU-GEMM/ BFU-E/CFU-M shift from the multipotent/erythroid/monocytic to the granulocytic-neutrophilic differentiation program.

Retinoic Acid Downmodulates Erythroid Differentiation and GATA1 Expression in Purified Adult-Progenitor Culture

Abstract All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of interleukin-3 (IL-3) granulocyte- macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Ep) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS- liquid- suspension culture supplemented with saturating Ep level and low-dose IL-3/GM-CSF, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master GATA1 gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of GATA1 mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of GATA1 induction in the early stages of erythropoietic differentiation.

Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult-progenitor culture

All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of interleukin-3 (IL-3) granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Ep) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS- liquid-suspension culture supplemented with saturating Ep level and low-dose IL-3/GM-CSF, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master GATA1 gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of GATA1 mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of GATA1 induction in the early stages of erythropoietic differentiation.