Differentiation In Liquid Culture Research Articles

Age-Acquired Downregulation of Lmna Leads to Epigenetic Deregulation and Altered HSPC Function

Hematopoietic stem cells (HSCs) exhibit epigenetic reprogramming and decline in function with aging, and these changes may be predisposing mechanisms for development of clonal hematopoiesis and myeloid malignancies. Our recent study characterizing the epigenetic and transcriptional landscape of young (18-30 years) and aged (65-75 years) human CD34 +CD38 - bone marrow (BM) cells identified Lamin-A/C (LMNA) as one of the most downregulated genes with aging (7.9-fold, p=1.9x10 -13). LMNA encodes the nuclear lamina protein Lamin-A/C and is mutated in the premature aging disorder Hutchinson-Gilford Progeria Syndrome.To determine whether downregulation of LMNA contributes to the phenotype of human HSC aging, we knocked down LMNA (LMNA KD) using shRNA in young, mobilized peripheral blood CD34 + cells. With an average knockdown of 60%, we observed ~33% increase in myeloid colony forming potential (p<0.05). LMNA KD also impaired differentiation in liquid culture as determined by persistence of CD34 expression in twice as many cells as controls (p<0.0001) while induction of myeloid CD11b expression was reduced by ~29% (p<0.05), as well as a trend to reduced erythroid (CD71 +GYPA +) differentiation. To investigate the effects of Lmna loss-of-function in vivo, we conditionally deleted Lmna in mice using Vav-Cre. Loss of Lmna (Lmna KO) had no effect on steady state hematopoiesis in young (10 weeks) or middle-aged (14 months) knockout mice. However, Lmna KO BM cells from middle-aged mice generated 2-fold more total colonies than wild-type, floxed controls (WT=126 vs KO=292, p<0.01).Given that nuclear architecture provides a layer of epigenetic regulation through chromatin-lamina interactions, we investigated how the epigenome changes in the context of LMNA deficiency. By super-resolution microscopy, we observed loss of LMNA localization from the nuclear periphery in LMNA KD human CD34 +cells (p<0.05). Since lamina-associated domains generally contain regions of repressed chromatin, we stained for histone H3K9me2 and observed that LMNA KD cells displayed increased scattering throughout the nucleus without a change in its density. Next, we performed ATAC-seq and identified 603 open chromatin sites showing changes in accessibility (FDR<0.1). Over 73% of these sites show reduced accessibility and were associated with genes showing reduced expression by RNA-seq. Gene ontology analysis of these sites identified genes associated with heme biosynthetic and cell differentiation processes (FDR<0.005). Moreover, gene set enrichment analysis of the RNA-seq confirmed that downregulated genes were enriched for genes downregulated in our previously reported CD34 + aging signature (NES=-1.46, FDR=0.001). To determine whether LMNA KD alters the epigenome, we performed ChIP-seq for active (H3K4me3, H3K27ac) and repressive (H3K9me2) histone marks. LMNA KD resulted in significant reduction of 36% of H3K27ac peaks, which mimics our previous observation of marked age-related losses of H3K27ac. Most differential peaks localized to gene promoters (50%) and introns (34%) and include genes involved in nuclear pore organization and gene silencing (FDR<0.001). Notably, for peaks located at intergenic regions, >35% overlapped with active enhancers we reported as lost with aging and were associated with genes involved in regulation of p38 MAPK cascade (enrichment=2.34E -5), an important signaling pathway regulating proliferation and differentiation of HSCs and leukemic cells.Taken together, LMNA deficiency recapitulates features of aging at the functional and epigenetic level. LMNA KD in young, human CD34 + cells impaired their differentiation while increasing their colony forming potential. Similarly in middle-aged mice, LMNA KO BM cells showed increased colony forming potential. Genomic changes induced by LMNA deficiency include reduced accessibility and gene transcription, accompanied by changes in localization and occupancy of histone H3K9me2 and H3K27ac, respectively. These epigenetic changes affect genes regulating differentiation and signaling pathways. Thus, we have demonstrated that in addition to its structural role, LMNA also contributes to chromatin regulation of hematopoietic pathways important for normal CD34 + function. DisclosuresNo relevant conflicts of interest to declare.

A Calreticulin Neoepitope-Directed Monoclonal Antibody Can Overcome JAK Inhibitor Resistance and Block TPO-Independent Megakaryocyte Differentation

Introduction: Mutations within the gene encoding calreticulin (CALR) are the second most common genetic aberration associated with primary myelofibrosis (PMF), observed in 70% of non-JAK2 V617F cases. Importantly, patients with CALR mutations do not effectively respond to JAK inhibitor therapy and no mutation specific therapy is currently in use. Virtually all CALR mutations identified in PMF are small insertions or deletions clustered within exon 9 leading to a neo-epitope peptide sequence which is thought to directly or indirectly activate the thrombopoietin receptor (TpoR) by a poorly defined mechanism. Here we engineered a neo-epitope specific monoclonal antobody that has striking biological activity against ruxolitinib persistent cells.Methods TF-1 TpoR cells expressing TpoR were supplemented with 20 ng/mL of TPO. Rats were immunised with a CALR mutant peptide coupled to KLH. Serum from the immunised rats was screened by enzyme linked immunoassay, to verify a strong titre to the peptide immunogen. Primary PMF CD34+ cells were cultured in StemCell Pro with human SCF, IL-6 and IL-9. NSG mice were used to for engraftment studies after 150 cGy irradiation.Results: We engineered a panel of rat monoclonal antibodies after immunization with a 30 amino acid peptide corresponding to the C-terminal mutant CALR neoepitope sequence with an extra cysteine residue. Clone 4D7 showed superior activity of detecting mutant but not wild type CALR protein with a binding affinity of 13.5 pM and dissociation constant of 1.53 nM as measured by I 125-Scatchard. Treatment with 4D7 resulted in a significant (5-7-fold) increase in the amount of full-length mutant CALR protein in conditioned media. 4D7 inhibited Tpo-independent cell growth over 6 days in TF-1 cells expressing MPL and mutant CALR at 2, 10 and 20 µg. 4D7 blocked constitutive factor-independent phospho-STAT5 and phospho-ERK after incubation exclusively in mutant CALR cells but not in TF-1 cells expressing TpoR alone and increased the sub-G 0 fraction was observed compared to IgG control (P = 0.001, n = 3 independent experiments) consistent with induction of an apoptotic response. We tested activity in purified primary CD34+ cells obtained from patients with CALR mutant myelofibrosis using two orthogonal assays: - (i) Tpo-independent megakaryocyte differentiation in liquid culture and (ii) Tpo-independent megakaryocyte colony formation on a collagen-based medium. 4 out of 4 patient samples that displayed robust Tpo-independent growth of CD41+CD61+ megakaryocyte progenitors showed inhibition by 4D7 of at least 50%. Similarly, we saw dramatic reduction in the absolute numbers of primary Tpo-independent megakaryocyte colonies cultured on collagen (colony-forming unit-mega) treated with 4D7 in multiple patient samples (decrease of 46%, P = 0.0001, Student's t-test, n = 4 independent patient samples) Importantly, secretion of mutant CALR protein was neither upregulated nor downregulated by ruxolitinib, indicating ruxolitinib is unlikely to alter mutant CALR trafficking in patients. 4D7 had strong inhibitory activity on cells that were resistant to ruxolitinib, in both liquid culture at 96 hours or colony formation. To test whether 4D7 could block mutant CALR-dependent proliferation in vivo, we developed two distinct xenograft models, a bone marrow engraftment model, which measures mutant CALR dependent proliferation in the bone marrow microenvironment, and a chloroma model, which mimics extravascular infiltration of mutant CALR leukaemia, by injection of TPO-independent TF-1 cells in NSG mice. In the bone marrow engraftment model 4D7 treatment (12 mg/kg twice weekly via intraperitoneal injection) lowered peripheral blood engraftment of human CD33 myeloid cells at 3 weeks, bone marrow engraftment and significantly prolonged survival compared to IgG control (P=0.004, HR=0.2). In the chloroma model, 4D7 treatment resulted in significant decrease in tumour growth measured at 3 weeks (P<0.01) and improved overall survival (P=0.02, HR=0.07) compared to IgG controlConclusion: Together, these results suggest an immunotherapeutic approach may have clinical utility CALR-driven myeloproliferative neoplasms and CALR mutant acute myeloid leukaemia, as well as activity in CALR mutant patients that develop resistance/persistence to ruxolitinib. DisclosuresRoss: Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Keros Therapeutics: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

2035 – AGE-RELATED DOWNREGULATION OF LMNA IMPACTS HUMAN HEMATOPOIETIC STEM CELL FUNCTION

Hematopoietic stem cells, essential for the production of blood cells, decline in function with aging. Among these changes with aging are increased frequency of mutations in epigenetic modifiers. We analyzed the epigenetic landscape of Lineage-CD34+CD38- human HSC-enriched fraction (HSCe) from young (18-30 years), middle-aged (45-55 years), and aged (65-75 years) individuals by comparing changes in gene expression by RNA sequencing and histone marks (H3K4me3, H3K4me1, H3K27ac, H3K27me3) by ChIP sequencing. Integrative analysis of the histone profiles by k-means clustering identified 12 clusters of genes defined by their specific pattern of age-associated epigenetic changes, which were accompanied by changes in gene expression affecting 1,133 genes between young and aged HSCe. The LMNA gene, which encodes the nuclear lamina protein Lamin A/C, was one of the most downregulated genes with aging (7.9-fold, p=1.9 × 10−13). This downregulation was mediated by loss of active histone marks H3K4me3 at the LMNA gene promoter, as well as loss of H3K27ac at two putative enhancer regions. ShRNA-mediated knockdown of LMNA in young, CD34+ hematopoietic stem and progenitor cells (HSPCs) resulted in an increase in myeloid colony formation (n=7 biological replicates with an average of 33% in total colony increase), consistent with the aging phenotype. Moreover, knockdown of LMNA in CD34+ cells impaired myeloid differentiation in liquid culture as determined by persistence of CD34 expression (n=5, p≤0.0001) with a delay in CD11b expression (n=5, p≤0.05). Initial results by STORM super resolution imaging show loss of LMNA leads to enlarged nuclear size and changes in chromatin localization. In conclusion, loss of LMNA recapitulates features of HSC aging, and restoring LMNA levels may be important for restoring normal function in aged HSCs.

Streptomyces Differentiation in Liquid Cultures as a Trigger of Secondary Metabolism.

Streptomyces is a diverse group of gram-positive microorganisms characterised by a complex developmental cycle. Streptomycetes produce a number of antibiotics and other bioactive compounds used in the clinic. Most screening campaigns looking for new bioactive molecules from actinomycetes have been performed empirically, e.g., without considering whether the bacteria are growing under the best developmental conditions for secondary metabolite production. These screening campaigns were extremely productive and discovered a number of new bioactive compounds during the so-called “golden age of antibiotics” (until the 1980s). However, at present, there is a worrying bottleneck in drug discovery, and new experimental approaches are needed to improve the screening of natural actinomycetes. Streptomycetes are still the most important natural source of antibiotics and other bioactive compounds. They harbour many cryptic secondary metabolite pathways not expressed under classical laboratory cultures. Here, we review the new strategies that are being explored to overcome current challenges in drug discovery. In particular, we focus on those aimed at improving the differentiation of the antibiotic-producing mycelium stage in the laboratory.

ArgR of Streptomyces coelicolor Is a Pleiotropic Transcriptional Regulator: Effect on the Transcriptome, Antibiotic Production, and Differentiation in Liquid Cultures.

ArgR is a well-characterized transcriptional repressor controlling the expression of arginine and pyrimidine biosynthetic genes in bacteria. In this work, the biological role of Streptomyces coelicolor ArgR was analyzed by comparing the transcriptomes of S. coelicolor ΔargR and its parental strain, S. coelicolor M145, at five different times over a 66-h period. The effect of S. coelicolor ArgR was more widespread than that of the orthologous protein of Escherichia coli, affecting the expression of 1544 genes along the microarray time series. This S. coelicolor regulator repressed the expression of arginine and pyrimidine biosynthetic genes, but it also modulated the expression of genes not previously described to be regulated by ArgR: genes involved in nitrogen metabolism and nitrate utilization; the act, red, and cpk genes for antibiotic production; genes for the synthesis of the osmotic stress protector ectoine; genes related to hydrophobic cover formation and sporulation (chaplins, rodlins, ramR, and whi genes); all the cwg genes encoding proteins for glycan cell wall biosynthesis; and genes involved in gas vesicle formation. Many of these genes contain ARG boxes for ArgR binding. ArgR binding to seven new ARG boxes, located upstream or near the ectA-ectB, afsS, afsR, glnR, and redH genes, was tested by DNA band-shift assays. These data and those of previously assayed fragments permitted the construction of an improved model of the ArgR binding site. Interestingly, the overexpression of sporulation genes observed in the ΔargR mutant in our culture conditions correlated with a sporulation-like process, an uncommon phenotype.

The APC/C Coactivator Cdh1 Controls Self-Renewal and Differentiation of Human and Murine HSPCs

Introduction: The balance between differentiation and self-renewal in hematopoietic stem and progenitor cells (HSPCs) is crucial for homeostasis and lifelong blood cell production. Differentiation is predominantly initiated in the G1 phase of the cell cycle when the E3 ligase anaphase-promoting complex or cyclosome (APC/C) is highly active. Its coactivator Cdh1 determines substrate specificity and mediates proteasomal degradation. Relevant target proteins are associated with cell fate decisions in G1/G0, and there is growing evidence that Cdh1 is an important regulator of differentiation. While this has already been demonstrated in neurons, muscle cells or osteoblasts, little is known about the role of APC/CCdh1 in hematopoiesis. Here we report on the function of Cdh1 in human and murine HSPCs in vitro and in vivo.Methods: Human CD34+ cells from the peripheral blood of G-CSF mobilized donors were exposed to different cytokine combinations and gains or losses of surface marker expression during cell division were determined. By using the established culture conditions Cdh1 expression was detected in distinct hematopoietic lineages and developmental states. CD34+ cells were transduced with a lentivirus to deplete Cdh1 by stably expressing shRNA and was then used for in vitro differentiation in liquid culture or CFU assay. In a second miR-based RNAi approach murine BM cells were depleted of Cdh1 and used for competitive transplantation assays. Complementary xenotransplantation of human Cdh1-depleted CD34+cells was carried out with NSG mice.Results: The stimulation of freshly thawed CD34+ cells with cytokines led to cell cycle entry and proliferation. Self-renewing cells preserved CD34 expression for up to 7 cell divisions with a low proliferation rate. In contrast, during granulopoiesis and erythropoiesis cells divided more frequently with rapid down-regulation of CD34. Cdh1 expression was tightly connected to differentiation status and proliferation properties. In vitro cultured CD34+ cellsand those from BM of healthy human donors showed the highest Cdh1 level compared to moderate or low expression in lymphoid and myeloid cells. Cdh1 is highly expressed at the transcriptional and translational level during both self-renewal and also when cells were directed toward erythroid differentiation. Therefore, high Cdh1 expression is characteristic of immature hematopoietic cells and differentiating precursors. The knockdown of Cdh1 (Cdh1-kd) did not affect proliferation or viability as detected by CFSE staining and measuring the cell cycle length via live-cell imaging. However, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with a lower frequency of glycophorin A+ cells. The functional relevance of Cdh1 depletion was verified in CFU assays. Cells with Cdh1-kd formed fewer primary colonies but significantly more secondary colonies, indicating a preference for self-renewal over differentiation. After competitive transplantation Cdh1-depleted murine BM cells showed a significant enhancement in the repopulation of PB, BM and spleen at week 3, while there was no change in cell cycle properties. However, after 8 weeks chimerism in each of the compartments was reduced to that of the control cells. Accordingly, higher LK and LSK frequencies supported the engraftment of Cdh1-depleted cells at week 3, but there was a significant decrease at week 8 compared to control cells, suggestive of stem cell exhaustion. The Cdh1 level also affected cell differentiation in vivo. After 8 weeks the population of B cells (B220+) was increased in transplanted Cdh1-kd cells and the frequency of mature granulocytes (CD11b+ Gr1high) was reduced. Consistently, human Cdh1-depleted CD34+ cells engrafted to a much higher degree in the murine BM 8 and 12 weeks after xenotransplantation, as shown by a higher frequency of human CD45+ cells. Moreover, the increase of human CD19+ B cells with Cdh1-kd confirmed the results of the competitive transplantation.Conclusions: Loss of the APC/C coactivator Cdh1 supports repopulation of murine HSPCs after transplantation with a lymphoid-biased differentiation, and was confirmed in xenotranplantation experiments. In the long-term, Cdh1 loss led to exhaustion of primitive LK and LSK population, highlighting the role of Cdh1 as a critical regulator of HSPC self-renewal and differentiation. DisclosuresEngelhardt:Janssen: Research Funding; Amgen: Research Funding; MSD: Research Funding; Celgene: Research Funding.

Self-Renewal and Differentiation in Hematopoietic Stem and Progenitor Cells Is Controlled By the APC/C Coactivator Cdh1

Introduction: Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously renew the hematopoietic system by differentiation into mature blood cells. The process of differentiation is predominantly initiated in G1 phase of the cell cycle when stem cells leave their quiescent state. During G1 the anaphase-promoting complex or cyclosome (APC/C) associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate proliferation. In addition, Cdh1 has been shown to control terminal differentiation in neurons, muscle cells or osteoblasts. Here we show that Cdh1 is also a critical regulator of human HSPC differentiation and self-renewal.Methods: Human CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokine combinations. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with flow cytometric detection of CD34 expression. The knockdown and overexpression of Cdh1 was achieved by lentiviral delivery of suitable vectors into target cells. After cell sorting transduced (GFP+) CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice.Results: G-CSF mobilized CD34+ cells showed effective differentiation into granulocytes (SCF, G-CSF), erythrocytes (SCF, EPO) or extended self-renewal (SCF, TPO, Flt3-L) when stimulated in vitro. The differentiation was characterized by a fast downregulation of Cdh1 on protein level, while Cdh1 remained expressed under self-renewal conditions. A detailed analysis of different subsets, both in vitro and in vivo, showed high Cdh1 level in CD34+ cells and low expression in myeloid cells. Analysis of proliferation revealed lowest division rates during self-renewal, accompanied by higher frequency of CD34+ cells. The fastest proliferation was found after induction of erythropoiesis. These experiments also showed a more rapid decrease of HSPCs' colony-forming ability and of CD34+ cells during granulopoiesis after 2-3 cell divisions in contrast to a moderate decline under self-renewal conditions. The depletion of Cdh1 (Cdh1-kd) had no effect on total cell numbers or proliferation detected by CFSE during differentiation and self-renewal, but showed an increase in S phase cells. These results were confirmed at the single cell level by measuring the cell cycle length of individual cells. Independent of cell cycle regulation, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of Glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies compared to control cells. These results suggest that the majority of cells reside in a more undifferentiated state due to Cdh1-kd. The overexpression of Cdh1 showed reversed results with less S phase cells and tendency to increased differentiation in liquid culture and CFU assays. To further validate our results in vivo, we have established a NSG xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Preliminary data in murine hematopoiesis support our hypothesis showing enhanced PB chimerism upon Cdh1-kd. Looking for a mediator of these effects, we found the Cdh1 target protein TRRAP, a cofactor of many HAT complexes, increased upon Cdh1-kd under self-renewal conditions. We use currently RT-qPCR to determine, if this is caused by a transcriptional or post-translational mechanism.Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells, represses erythropoiesis in vitro and facilitates engraftment capacity and B cell development of human HSPCs in vivo.This work was supported by Josè Carreras Leukemia Foundation grant DCJLS R10/14 (to ME+RW) DisclosuresEwerth:Josè Carreras Leukemia Foundation: Research Funding. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Unexplained Anemia In The Elderly Is Due To An Intrinsic Defect In Hematopoietic Stem Cells

In about 1/3 of older anemic adults, no clinical etiology can be found and this group has been labeled unexplained anemia in the elderly (UAE). UAE has been associated with poor outcomes, including decreased functional status, increased rate of hospitalization, and increased mortality. We hypothesize that intrinsic functional impairment of hematopoietic stem and progenitor cells is a significant contributor to the pathogenesis of UAE. Hematopoietic stem cells (HSCs) reside at the top of the hematopoietic hierarchy and can differentiate, via committed downstream progenitors, into all the mature cells of the hematopoietic system. Human erythroid development proceeds through a set of oligopotent progenitors that can be isolated by immunophenotypic markers: HSCs (Lin-CD34+CD38-CD90+CD45RA-) give rise to multipotent progenitors (MPPs), which give rise to common myeloid progenitors (CMPs), which give rise to granulocyte-macrophage progenitors (GMPs) and megakaryocyte-erythrocyte progenitors (MEPs; Lin-CD34+CD38+CD123-CD45RA-). In addition, MEPs can be further fractionated into erythroid-biased MEPs (E-MEPs; Lin-CD34+CD38+CD123-CD45RA-CD71+CD105-), which give rise to erythroid-committed progenitors (EPs; Lin-CD34+CD38+CD123-CD45RA-CD71+CD105+), which subsequently differentiate into early erythroid precursors (pre-Es; CD34-CD71+GPA-). We compare immunophenotypic hematopoietic stem, progenitor, and erythroid precursor populations obtained from hematologically normal young (25 samples; age 20-35), normal elderly (11 samples; age 65+), and UAE (9 samples; age 65+) bone marrow samples. We find that UAE patients contain increased frequencies of MEP compared to normal (1.4-fold; p<0.006). However, there are fewer E-MEPs in UAE patients, suggesting that MEPs from UAE patients do not readily differentiate towards the erythroid lineage (p<.04). Interestingly, we find that one cohort (n = 3) of UAE patients exhibits an expanded EP population and a reduced pre-E population while another cohort (n =4) exhibits the converse. We hypothesize that UAE patients with an expanded EP population have a defect in the ability of EPs to differentiate into downstream pre-Es, whereas UAE patients with a reduced EP population have a defect in the ability of E-MEPs to differentiate into downstream EPs. Functionally, HSCs from UAE patients produce relatively more granulocyte-monocyte (CFU-GM) colonies and fewer erythroid (CFU-E and BFU-E) colonies compared to HSCs from normal young and elderly samples (p<0.008). Similarly, CMPs and MEPs from UAE patients yield skewed distributions of myeloid-erythroid colonies when plated in methylcellulose, significantly favoring production of CFU-GM colonies over CFU-E and BFU-E colonies (CMP p<0.003; MEP p<0.02). Under conditions for erythroid differentiation in liquid culture, E-MEPs from UAE patients with a reduced EP population, but not from patients with an expanded EP population, produce fewer mature erythroid (GPA+) cells compared to normal (p<0.03). EPs and pre-Es from both UAE patient cohorts display defective mature erythroid differentiation potential (EP p<0.05; pre-E p<0.05). We find a functional impairment in the ability of UAE HSCs to differentiate into more mature erythroid forms in both methylcellulose and liquid culture. Thus, we hypothesize that the underlying pathophysiology in UAE is at least in part due to an intrinsic HSC defect. Additionally, although the pathophysiology of UAE remains obscure, we find that UAE can be divided into two surprisingly uniform hematopoietic progenitor phenotypes, suggesting two possible underlying impairments in hematopoiesis. Better understanding of underlying pathophysiology is the first step leading towards rational targeted therapeutic interventions. Disclosures:No relevant conflicts of interest to declare.

Elevated Levels Of γ-Globin Gene Promoter 5-Hydroxymethylcytosine are Associated With Increased DNA Hypomethylation and HbF Expression

DNA methylation of the γ-globin gene promoter represses γ-globin expression in adult-stage erythroid cells while high level γ-globin expression in fetal liver erythroid cells is associated with DNA hypomethylation. Previously we showed that DNA demethylation of the γ-globin gene promoter during fetal liver erythroid differentiation is responsible for the nearly complete loss of DNA methylation, while much more limited DNA demethylation during adult bone marrow (BM) erythroid differentiation maintains a relatively high level of γ-globin promoter DNA methylation (Singh et al Exp Hematol 35:48, 2007). As recent studies have shown the importance of 5-hydroxymethylcytosine (5-hmC) as an intermediate in active and passive mechanisms of DNA demethylation that alter epigenetic modifications regulating development and hematopoietic differentiation, experiments were performed to 1) investigate the hypothesis that DNA demethylation of the γ-globin promoter during erythroid differentiation involves 5-hmC, and 2) evaluate the role of 5-hmC in γ-globin gene expression. Levels of 5-hmC and 5-methylcytosine (5-mC) located at a CpG residue within the context of a HpaII site within the 5' γ-globin promoter were measured in 1) baboon BM cells enriched for different stages of erythroid differentiation, and 2) CD34+ BM-derived erythroid progenitors expressing high levels of γ-globin grown in liquid culture or expressing low levels of γ-globin in co-culture with the AFT024 cell line. Analysis of BM cells showed that CD117+CD36+ BM cells enriched in clonogenic late BFUe/CFUe had nearly 3 fold higher levels of γ-globin promoter 5-hmC (6.91+1.41%) compared to BM-derived erythroid precursors (2.57+0.75%; p<0.0001). In erythroid precursors expressing low levels of HbF derived from CD34+ BM cells grown in co-culture with the AFT024 murine fetal liver cell line, the levels of γ-globin promoter 5-hmC (1.72+1.19%) and 5-mC (64.84+9.22%) were not significantly different from BM-derived erythroid precursors. In contrast, the level of γ-globin promoter 5-hmC in erythroid precursors derived from CD34+ BM cells grown in liquid cultures expressing elevated levels of HbF was significantly higher (6.18+1.35%) than terminal erythroid precursors from either adult BM (p<.0001) or AFT024 co-cultures (1.72+1.19%; p<.0001) but was not significantly different than the level in CD117+CD36+ BM cells. Levels of γ-globin promoter 5-hmC were similar in erythroid precursors from liquid cultures on d7 (5.57+1.24%), d11 (6.13+1.02%), d14 (6.73+1.74%), and more primitive d7 gly- basophilic erythroblasts (6.21+1.38%). The level of DNA methylation (5-mC) was significantly less in erythroid precursors derived from liquid cultures (40.37+14.33%) compared to erythroid precursors derived from adult BM (63.10+7.72%; p<0.0005), AFT024 co-cultures (64.84+9.22%; p<0.001) and CD117+CD36+ BM cells (67.71+7.45%; p<0.002). Reduced levels of 5-mC were observed in erythroid precursors from liquid cultures on d14 (34.87+14.67%) compared to d7 (48.64+15.56%; p<0.055) suggesting that the γ-globin gene is progressively demethylated during erythroid differentiation in liquid culture. We conclude that γ-globin promoter 5-hmC levels are modulated during adult BM erythroid differentiation with 3 fold higher levels in CD117+CD36+ cells enriched in late BFUe/CFUe compared to erythroid precursors. Similar levels of γ-globin promoter 5-hmC, 5-mC, and HbF are observed in adult BM erythroid precursors and erythroid precursors derived from AFT024 co-cultures. In contrast, high levels of levels of γ-globin promoter 5-hmC, similar to levels in CD117+CD36+ BM cells, are sustained in erythroid precursors derived from liquid cultures of CD34+ BM cells and are associated with decreased γ-globin promoter 5-mC and increased HbF. Supplementation of cultures with ascorbic acid, a co-factor of the TET oxygenases that catalyze 5-hmC, reduced levels of γ-globin promoter 5-mC (20.94+9.77%) compared to controls (51.24+14.61%; p<.025) and increased γ-globin expression. These results support the hypothesis that DNA demethylation of the γ-globin promoter during erythroid differentiation, resulting in high HbF expression, occurs through a 5-hmC-mediated mechanism subject to developmental regulation by factors in the micro-environment. Disclosures:No relevant conflicts of interest to declare.

Sotatercept, an Activin Receptor IIa Ligand Trap, Acts Through Bone Marrow Accessory Cells to Promote Late-Stage Erythropoiesis and a Rapid Induction of Red Blood Cell Number and Hemoglobin

Abstract 372The regulation of erythropoiesis requires stem cell factor and erythropoietin (EPO) for the proliferation and survival of erythroid progenitor and early precursor cells. While recombinant EPO is widely used for treating various types of anemia, it often lacks efficacy in cases of anemia due to ineffective erythropoiesis in which immature erythroid precursors undergo apoptosis. Thus, there is an need for new therapies to treat the later stages of erythropoiesis.Members of the transforming growth factor beta (TGFβ) superfamily have been studied as potential regulators of erythropoiesis, iron regulation and globin expression. Sotatercept (ACE-011), a recombinant fusion protein consisting of the extracellular domain of the human activin receptor IIA (ActRIIA) linked to the human immunoglobulin G1 (IgG1) Fc domain, is a ligand trap which binds a number of TGFβ superfamily ligands including activin A, activin B, growth differentiation factor-11 (GDF-11) and bone morphogenetic protein-10 (BMP-10). Administration of sotatercept led to substantial increases in red cell number and hemoglobin in human subjects, but the mechanism is not fully understood. We utilized both mouse in vivo and human in vitro models to investigate the mechanism of sotatercept in promoting erythropoiesis.In order to compare the effects of RAP-011 (the murine version of sotatercept) to EPO on red blood cell (RBC) parameters, C57/Bl mice were dosed with RAP-011, EPO or control vehicle. RAP-011-treated mice had a rapid and statistically significant increase in hematocrit, hemoglobin, and RBC number in less than 72-hours. As rapidly as 24 hours after treatment, RAP-011 induced a significant increase in RNA-negative, enucleated cells in the bone marrow (BM). RAP-011 also rapidly increased BM BFU-e and CFU-e erythroid progenitors, while EPO was more effective on spleen-derived progenitors. These data suggest that RAP-011 acts primarily on both bone marrow progenitor cells and late erythroblasts to promote erythropoiesis.In order to investigate the cellular mechanism by which RAP-011 increases red blood cell parameters, we conducted a series of in vitro experiments and found no evidence to support direct effects of RAP-011 on human CD34+ cells assessed in colony formation assays and in erythroid differentiation in liquid culture. As both clinical and pharmacological findings point to a clear role for RAP-011 in stimulating RBC parameters, we hypothesized that RAP-011 effects may be mediated by accessory cells in the BM microenvironment. Human CD36+ cells, which are highly enriched for erythroid progenitors, were co-cultured with long-term BM cultures and erythroid differentiation was assessed following 6 days of culture in EPO (2U/mL)-supplemented media. At day 6 the output of these cultures was predominantly characterized as EryA (∼basophilic erythroblast) but with the addition of RAP-011 (50μM), a significant fraction of CD36+ cells matured into EryB/C cells (polychromatic/orthochromatic erythroblasts), suggesting that factors produced by BM accessory cells mediate RAP-011 erythropoietic effects and that, in contrast to EPO, RAP-011 may play a role in the latter stages of erythroblast maturation. To identify cytokines that may mediate RAP-011 effects, CD36+ cells were treated with several activin receptor IIA ligands. GDF-11 treatment significantly decreased proliferation of GPA+ cells during the differentiation process and RAP-011 effectively reversed this effect, but had no consequence on untreated cells. These data suggest that GDF-11 may mediate the erythroipoietic stimulatory effects of RAP-011.In summary, RAP-011 induced a rapid increase in RBC parameters in mice (reflected in the number of enucleated cells found in the bone marrow), likely mediated by BM accessory cells. Our data also suggest that effects of sotatercept may be mediated at least partly by GDF-11, acting as a potential negative regulator of the terminal stages of erythropoiesis. The ability of sotatercept to reverse this inhibition would lead to a rapid release of terminal erythroid cells into the circulation. These data support the rationale to develop sotatercept for the treatment of anemia and ineffective erythropoiesis, especially in patients who may not respond to EPO. Disclosures:Carrancio:Celgene Corporation: Employment. Markovics:Celgene Corporation: Employment. Wong:Celgene Corporation: Employment. Leisten:Celgene Corporation: Employment. Groza:Celgene Corporation: Employment. Raymon:Celgene Corporation: Employment. Heise:Celgene Corporation: Employment. Chopra:Celgene Corp: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment. Sung:Celgene Corporation: Employment.

HSP27 controls GATA-1 protein level during erythroid cell differentiation

AbstractHeat shock protein 27 (HSP27) is a chaperone whose cellular expression increases in response to various stresses and protects the cell either by inhibiting apoptotic cell death or by promoting the ubiquitination and proteasomal degradation of specific proteins. Here, we show that globin transcription factor 1 (GATA-1) is a client protein of HSP27. In 2 models of erythroid differentiation; that is, in the human erythroleukemia cell line, K562 induced to differentiate into erythroid cells on hemin exposure and CD34+ human cells ex vivo driven to erythroid differentiation in liquid culture, depletion of HSP27 provokes an accumulation of GATA-1 and impairs terminal maturation. More specifically, we demonstrate that, in the late stages of the erythroid differentiation program, HSP27 is phosphorylated in a p38-dependent manner, enters the nucleus, binds to GATA-1, and induces its ubiquitination and proteasomal degradation, provided that the transcription factor is acetylated. We conclude that HSP27 plays a role in the fine-tuning of terminal erythroid differentiation through regulation of GATA-1 content and activity.

Cysteinyl-leukotriene type 1 receptors transduce a critical signal for the up-regulation of eosinophilopoiesis by interleukin-13 and eotaxin in murine bone marrow

IL-13 and eotaxin play important, inter-related roles in asthma models. In the lungs, CysLT, produced by the 5-LO-LTC4S pathway, mediate some local responses to IL-13 and eotaxin; in bone marrow, CysLT enhance IL-5-dependent eosinophil differentiation. We examined the effects of IL-13 and eotaxin on eosinophil differentiation. Semi-solid or liquid cultures were established from murine bone marrow with GM-CSF or IL-5, respectively, and the effects of IL-13, eotaxin, or CysLT on eosinophil colony formation and on eosinophil differentiation in liquid culture were evaluated, in the absence or presence of: a) the 5-LO inhibitor zileuton, the FLAP inhibitor MK886, or the CysLT1R antagonists, montelukast and MK571; b) mutations that inactivate 5-LO, LTC4S, or CysLT1R; and c) neutralizing mAb against eotaxin and its CCR3 receptor. Both cytokines enhanced GM-CSF-dependent eosinophil colony formation and IL-5-stimulated eosinophil differentiation. Although IL-13 did not induce eotaxin production, its effects were abolished by anti-eotaxin and anti-CCR3 antibodies, suggesting up-regulation by IL-13 of responses to endogenous eotaxin. Anti-CCR3 blocked eotaxin completely. The effects of both cytokines were prevented by zileuton, MK886, montelukast, and MK571, as well as by inactivation of the genes coding for 5-LO, LTC4S, and CysLT1R. In the absence of either cytokine, these treatments or mutations had no effect. These findings provide evidence for: a) a novel role of eotaxin and IL-13 in regulating eosinophilopoiesis; and b) a role for CysLTRs in bone marrow cells in transducing cytokine regulatory signals.

X-Linked Clonality Analysis of Human Embryonic Stem Cells ( hESC ) Before and after induction of Hematopoietic Differentiation

Human ES cell lines represent an invaluable tool of research for the study of normal and pathological development. In this study we asked whether the human ES cell lines currently used for research purposes are derived from single outgrowths using X-linked clonality analysis. Amongst the cell lines tested, the female H9 cell line was informative at the Humara (human androgen receptor) locus by the presence of two CAG repeats allowing distinction of paternal (Xp) and maternal (Xm) X chromosomes. Clonal analysis was performed upon Hpa II digestion of DNA purified from total H9 cells (passages 27–61), single H9 cell clones generated from single cell cloning, single embryoid bodies (EB) (day 4, day 14) as well as from single hematopoietic progenitors. The same analysis was performed after induction of hematopoietic differentiation in liquid cultures in the presence of hematopoietic permissive conditions (co-culture in the presence of OP-9 cell line). Clonality was evaluated by calculating the Relative Corrected Index (RCI) which is the ratio of the intensity of peaks generated by Hpa II-digested and undigested DNA. In several experiments, all passages of H9 cell lines examined ( p27, p28, p31, p61) were found to exhibit a monoclonal pattern with disappearance of the same allele A upon Hpa II digestion (RCI > 10). 13 clonal cultures of H9 obtained by single cell cloning exhibited also the same clonal pattern with digestion of the same allele and RCI values between 6–14. Similarly, DNA purified from EB's at different stages was found to be clonal. In DNA obtained from 75 individual hematopoietic progenitors (CFU-GM, CFU-G), clonal analysis revealed the same clonal pattern in each, with disappearance of the same allele A. However, clonal analysis performed in 10 hematopoietic colonies, revealed unexpectedly, an equal digestion of Humara alleles with RCI < 2 (polyclonal pattern). Finally, in two experiments, hematopoietic cells recovered from liquid cultures exhibited an absolute clonal pattern (Total disappearance of the same allele A). Interestingly, quantification of X-Inactivation Specific Transcript (XIST) RNA in H9 and day14 EB's by RT-PCR did not show any evidence of XIST expression both before and after differentiation. Altogether, our data suggest that XCI has already been initiated in undifferentiated H9 cells and this inactivation is maintained, at least in the Humara locus in the absence of XIST. The monoclonal pattern of the cell line can be due to the fact that H9 ES cell line might have arisen from a limited number of stem cells having undergone XCI at a very early stage, with maintenance of the methylation status at this locus during mesodermal and hematopoietic commitment. Conversely, hematopoietic differentiation might occur from several mesodermal progenitors having inactivated the same X chromosome in H9 at the Humara locus. Despite the fact that hematopoietic progenitors and differentiated hematopoietic cells originating from H9 exhibited also a clonal pattern, a partial reactivation of the inactive X chromosome during hematopoietic differentiation could occur, and may explain the “polyclonal” pattern obtained in 10 CFU-GM analyzed. Overall, X-linked clonality testing could be an important issue before considering the potential clinical applications, in all female hES cell lines.

Autologous Stem Cell Transplantation (ASCT) for AIDS-Related Lymphomas (ARL) and the Potential Role of HIV-Resistant Stem Cells.

Background: Optimal therapy of ARL relies on both effective anti-tumor chemotherapy and successful control of the underlying HIV infection. Management of HIV using HAART has been hampered by patient non-compliance with complex regimens, drug resistance and ongoing low level viral replication. Multiplexed RNA based anti-HIV gene transfer strategies to confer intrinsic cellular resistance may help circumvent these problems. Autologous stem cell transplantation for ARL is an ideal clinical platform for delivery of SiRNA transduced stem cells as combining gene transfer strategy with high dose antilymphoma therapy could provide control of both the HIV infection and the ARL.Methods: Gene transfer - anti-HIV RNA elements, including short hairpin RNA (shRNA) targeted to HIV tat/rev, a TAR-specific decoy sequence, and a ribozyme targeted to CCR5 were combined into a lentivirus vector (LV, rHIV7-shI-TAR-CCR5RZ). Using LV transduction methods, these anti-HIV RNAs were delivered into CD34+ hematopoietic progenitor cells (HPC).Results: Preclinical vector development - LV transduction allowed differentiation in liquid culture and in a SCID-hu model which produced macrophage and T cell progeny that were resistant to the HIV virus. Although HIV resistance can be induced in vitro with single anti-HIV shRNAs, no resistance was found in multiply passaged HIV in rHIV7-shI-TAR-CCR5RZ-transduced cells. In addition, cells were analyzed by microarray for mRNA and miRNA alterations and no significant changes were noted between CD34 transduced and untransduced cells. Copy number in transduced cells was 1–2/cell, and integration site analysis localized to transcriptionally active sites, usually away from terminal portions of gene sequences. This vector is proposed for use in ASCT for ARL.Update of ASCT in ARL: Between 1998 and 2006, 28 patients with high-risk ARL underwent ASCT at the City of Hope. Conditioning regimens included CBV (cyclophosphamide (Cy)100mg/kg, Carmustine150 mg/m2, VP16 60mg/kg) in 24 and FTBI/Cy/VP16 in 4. All patients engrafted, median time to ANC>500 was 11 days (range, 8–19). Regimen related toxicities included grade 3–4 hepatic toxicity n=3 and interstitial pneumonitis n=2. OI's included PCP pneumonia n=2, CMV infection (2 viremias, 1 retinitis) and 2 cases of VZV. Therapy-related MDS was seen in one patient who ultimately died of MDS while in remission from his ARL. Median HIV viral load (VL) at ASCT was 6113 gc/ml with 22 having an undetectable VL. At two years only 9 pts had an undetectable VL. Median CD4 count at ASCT was 164 (range 25–1064), this rose to 263 (95–1164) at two years post ASCT. With a median f/u of 41 months, 2yr OS is 78% (95% CI 63–87) and PFS 78% (95% CI 63–87).Conclusions: ASCT can lead to durable remission for ARL. The fluctuation in VL seen post ASCT reflects the natural history of HIV infection and limitations of current antiviral therapy. Ultimately the system of gene transfer outlined above combined with ASCT as part of our next generation clinical trial could provide the optimal chance of cure for pts with high-risk ARL by offering both effective antilymphoma and anti-HIV therapy.

Functional analysis of leukemia-associated PTPN11 mutations in primary hematopoietic cells

PTPN11 encodes the protein tyrosine phosphatase SHP-2, which relays signals from growth factor receptors to Ras and other effectors. Germline PTPN11 mutations underlie about 50% of Noonan syndrome (NS), a developmental disorder that is associated with an elevated risk of juvenile myelomonocytic leukemia (JMML). Somatic PTPN11 mutations were recently identified in about 35% of patients with JMML; these mutations introduce amino acid substitutions that are largely distinct from those found in NS. We assessed the functional consequences of leukemia-associated PTPN11 mutations in murine hematopoietic cells. Expressing an E76K SHP-2 protein induced a hypersensitive pattern of granulocyte-macrophage colony-forming unit (CFU-GM) colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) that was dependent on SHP-2 catalytic activity. E76K SHP-2 expression also enhanced the growth of immature progenitor cells with high replating potential, perturbed erythroid growth, and impaired normal differentiation in liquid cultures. In addition, leukemia-associated SHP-2 mutations conferred a stronger phenotype than a germline mutation found in patients with NS. Mutant SHP-2 proteins induce aberrant growth in multiple hematopoietic compartments, which supports a primary role of hyperactive Ras in the pathogenesis of JMML.

Purified unfractionated G-CSF/chemotherapy mobilized CD34+ peripheral blood progenitors and not bone marrow CD34+ progenitors undergo selective erythroid differentiation in liquid culture in the presence of erythropoietin and stem cell factor.

A combination of erythropoietin (EPO) plus stem cell factor (SCF) drove purified unfractionated granulocyte colony stimulating factor (G-CSF)/chemotherapy mobilized peripheral blood CD34+ cells to selective erythroid differentiation in liquid culture with an average 28-fold increase in the total cell number after 21 d. From day 6 of culture cytologic and cytofluorimetric characterization revealed that cultured cells belonged to the erythroid lineage with a gradual wave of maturation along the erythroid pathway to terminal cells. A similar pattern of erythroid differentiation was observed when the same peripheral blood CD34+ cells were culture with EPO plus SCF in serum-free medium. This cytokine combination produced selective erythroid differentiation with the complete exhaustion of the clonogenic potential on day 21. In parallel experiments the same circulating CD34+ cells underwent granulocytic/ monocytic differentiation in liquid culture in response to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and SCF, demonstrating that these CD34+ progenitors had intact pluripotent differentiating potential. Conversely, bone marrow CD34+ cells isolated from bone marrow allografts were unable to selectively differentiate along the erythroid pathway when they were exposed to EPO plus SCF combination. However, these cells maintained a greater number of colony forming cells on day 21 of culture compared to mobilized peripheral blood CD34+ cells. This model is a simple and reliable way to obtain selective erythroid differentiation of peripheral blood G-CSF/ chemotherapy mobilized CD34+ progenitor cells in liquid culture. The absence of cytokines such as GM-CSF and IL-3 in the culture medium permits studies on in vitro erythropoiesis without disturbance of prevalent myelopoiesis.

Expression and Extracellular Release of Transferrin Receptors During Peripheral Erythroid Progenitor Cell Differentiation in Liquid Culture

AbstractThe expression and extracellular release of transferrin receptor (TR) was investigated by in vitro model system of erythroid differentiation. Human peripheral blood mononuclear cells were cultured with interieukin-3 (IL-3) for 7 days, and with erythropoietin (EPO) for an additional 8 days. After EPO stimulation, IL-3-stimulated blastic cells were serially differentiated into mature erythrocytes. [3H]-thymidine incorporation of cultured cells increased linearly from day 0 to 5, followed by a decrease. Flow cytometric analysis showed an increase of TR expression from day 0 to 5, followed by a slight decrease. By metabolic labeling with [35S]methionine and immunoprecipitation, the cell lysate exhibited a 95-kD band corresponding to the intact TR on sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography at day 5, when polychromatic erythroblasts had their peak. The culture supernatant solubilized by tween-20 exhibited a 95-kD and an 85-kD band on days 5 and 8, which corresponded to the intact and the truncated forms of TR, respectively. The 95-kD band was more intense at day 5 than at day 8. The reverse transcriptase-polymerase chain reaction assay showed that the receptor-mRNA expression was parallel to receptor synthesis. Thus, the synthesis and expression of TR on erythrocytes is associated mainly with cell proliferation in the early phase, and with both cell proliferation and hemoglobin production in the middle to late phases of maturation. Concomitantly, the extracellular release of TR from erythrocytes occurs in the middle to late phases of maturation. These data suggest that polychromatic erythroblasts release soluble TR as both intact and truncated forms and may be an important source of serum TR implicated as an index for erythropoietic activity in the marrow.

Expression and extracellular release of transferrin receptors during peripheral erythroid progenitor cell differentiation in liquid culture

The expression and extracellular release of transferrin receptor (TR) was investigated by in vitro model system of erythroid differentiation. Human peripheral blood mononuclear cells were cultured with interleukin- 3 (IL-3) for 7 days, and with erythropoietin (EPO) for an additional 8 days. After EPO stimulation, IL-3-stimulated blastic cells were serially differentiated into mature erythrocytes. [3H]-thymidine incorporation of cultured cells increased linearly from day 0 to 5, followed by a decrease. Flow cytometric analysis showed an increase of TR expression from day 0 to 5, followed by a slight decrease. By metabolic labeling with [35S]methionine and immunoprecipitation, the cell lysate exhibited a 95-kD band corresponding to the intact TR on sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography at day 5, when polychromatic erythroblasts had their peak. The culture supernatant solubilized by tween-20 exhibited a 95-kD and an 85-kD band on days 5 and 8, which corresponded to the intact and the truncated forms of TR, respectively. The 95-kD band was more intense at day 5 than at day 8. The reverse transcriptase-polymerase chain reaction assay showed that the receptor- mRNA expression was parallel to receptor synthesis. Thus, the synthesis and expression of TR on erythrocytes is associated mainly with cell proliferation in the early phase, and with both cell proliferation and hemoglobin production in the middle to late phases of maturation. Concomitantly, the extracellular release of TR from erythrocytes occurs in the middle to late phases of maturation. These data suggest that polychromatic erythroblasts release soluble TR as both intact and truncated forms and may be an important source of serum TR implicated as an index for erythropoietic activity in the marrow.

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.