Differentiation Of Human CD34 Research Articles

Lenalidomide and pomalidomide modulate hematopoietic cell expansion and differentiation in the presence of MSC.

Cytopenia is a well-documented complication in the treatment of hematological malignancies with lenalidomide and pomalidomide. Although prior studies have highlighted direct effects on hematopoietic cells to explain this adverse effect, the involvement of hematopoietic-supportive stroma remains less understood. This study examined the effects of lenalidomide/pomalidomide on the expansion and differentiation of human CD34+ hematopoietic stem/progenitor cells (HSPCs) in vitro, in co-culture with human bone-marrow mesenchymal stromal/stem cells (MSCs). Our findings indicate that lenalidomide/pomalidomide increases the population of immature CD34+CD38- cells while decreasing the number of mature CD34+CD38+ cells, suggesting a mechanism that inhibits early HSPC maturation. This effect persisted across myeloid, megakaryocytic, and erythroid lineages, with MSCs playing a key role in preserving immature progenitors and inhibiting their differentiation. Furthermore, in myeloid differentiation assays augmented by granulocyte-colony stimulating factor, lenalidomide/pomalidomide not only enhanced the presence of CD34+ cells with mature myeloid markers such as CD11b but also reduced the populations lacking CD34 yet positive for these markers, irrespective of MSC presence. Thus, while MSCs support the presence of these immature cell populations, they simultaneously inhibit their maturation. This finding provides novel mechanistic insights into lenalidomide- and pomalidomide-induced cytopenia, and could guide therapeutic strategies for its mitigation.

Identification of a genomic DNA sequence that quantitatively modulates KLF1 transcription factor expression in differentiating human hematopoietic cells

The onset of erythropoiesis is under strict developmental control, with direct and indirect inputs influencing its derivation from the hematopoietic stem cell. A major regulator of this transition is KLF1/EKLF, a zinc finger transcription factor that plays a global role in all aspects of erythropoiesis. Here, we have identified a short, conserved enhancer element in KLF1 intron 1 that is important for establishing optimal levels of KLF1 in mouse and human cells. Chromatin accessibility of this site exhibits cell-type specificity and is under developmental control during the differentiation of human CD34+ cells towards the erythroid lineage. This site binds GATA1, SMAD1, TAL1, and ETV6. In vivo editing of this region in cell lines and primary cells reduces KLF1 expression quantitatively. However, we find that, similar to observations seen in pedigrees of families with KLF1 mutations, downstream effects are variable, suggesting that the global architecture of the site is buffered towards keeping the KLF1 genetic region in an active state. We propose that modification of intron 1 in both alleles is not equivalent to complete loss of function of one allele.

Abstract PO007: Role of PI3K pathway in reprogramming the tumor niche of angiosarcoma

Abstract Angiosarcoma (AS) is a highly aggressive, rare soft-tissue sarcoma that forms malignant blood vessels. Approximately half of patients have metastatic or unresectable disease with a median overall survival of less than 6 months, and tumor-related mortality is high. Inflamed tissue is frequently seen in angiosarcomas, and immune cells are key components that contribute to the landscape of the tumor microenvironment. Hemangiosarcoma (HSA) occurs commonly in companion dogs, and it shares clinical and morphological features with human AS. Recurrent mutations in TP53 and genes involved in PI3K pathway such as PIK3CA, and PIK3R1 were observed in both human AS and canine HSA, and the mutational landscape was associated with distinct molecular tumor subtypes; in particular, differing immunophenotypes. Recently, we have shown that human AS and canine HSA establish convergent transcriptional programs driven by angiogenic fusion genes and TP53 mutation. However, these vascular tumors are genomically complex, and molecular mechanisms that establish the tumor microenvironment are incompletely known. In this study, we analyzed RNA-seq transcriptomic data generated from human AS tissues (n=13) and canine HSA tissues (n=76) and HSA cell lines (n=11). The data showed that the tumors of both species enriched gene signatures associated with maintenance of hematopoietic stem cells. Then, we found that canine HSA cells supported expansion and differentiation of human CD34+ umbilical cord blood cells using long-term culture-initiating cell and colony-forming unit assays. We also developed xenograft models of canine HSA that facilitate myelopoiesis and macrophage infiltration in the tumor tissue. Our data showed that gene signatures of monocytes and naïve macrophages were detectable in canine HSA cells. In human ASs, macrophage-associated genes, in particular M2 macrophage signatures were significantly enriched, compared to normal tissues. Collectively, our data suggest that human AS and canine HSA have cell-autonomous capacity to govern hematopoietic progenitors and immune cells, potentially establishing the tumor immune niche. We engineered HSA cells to induce homozygous H1047R mutations in PIK3CA gene using CRISPR-Cas9 system. Our ongoing work is to determine if regulating PI3K pathway in the tumor cells contributes to the molecular programs that create the tumor niche of the vascular malignancy. Citation Format: Sophia Wenthe, Ashley J. Schulte, Mathew G. Angelos, Dan S. Kaufman, Jaime F. Modiano, Jong Hyuk Kim. Role of PI3K pathway in reprogramming the tumor niche of angiosarcoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO007.

AAV-Vectored Fms-Related Tyrosine Kinase 3 Ligand Inhibits CD34+ Progenitor Cell Engraftment in Humanized Mice.

Humanized mice have become useful animal models for HIV/AIDS. Since NOD.Cg-Prkdc scid Il2rgtm1Wjl/SzJ (NSG) mice allow the engraftment of primary human immune cells, we aim to determine the role of human Fms-related tyrosine kinase 3 ligand (hFlt3L), a major growth factor for dendritic cells (DCs), in regulating the differentiation of cord blood-derived CD34+ progenitor cells in this murine species. Soluble recombinant hFlt3L protein and AAV-vectored hFlt3L were administrated before or after human CD34+ progenitor cell transplantation, respectively. We then measured the peripheral levels of hFlt3L by ELISA. Meantime, reconstituted human immune cells were analyzed by flow cytometry over time. We found that without hFlt3L there were significantly increased types of human immune cells in NSG-huCD34 compared with NSG-huPBL mice but the frequency of human DCs remains low. Transient treatment with recombinant hFlt3L expanded human conventional CD1c+ and CD141+ DCs as well as plasmacytoid DCs in humanized NSG-huCD34 mice. Surprisingly, however, the prolonged in vivo expression of AAV-vectored hFlt3L resulted in significant suppression of total human CD34+ cell engraftment and differentiation. The suppression occurred within 2weeks when AAV-vectored hFlt3L was administered either before or after the transplantation of CD34+ progenitor cells, which was likely associated with the induction of murine myeloid-derived immune suppressive cells and reactive oxygen species in NSG-huCD34 mice. Sincechronic HIV-1 patients displayed significantly high levels of hFlt3L expression, our findings may have implication to explore the role of prolonged hFlt3L in regulating the differentiation of human CD34+ progenitor cells in both NSG-huCD34 mice and infected people. Graphical Abstract ᅟ.

Preclinical Development of a Hematopoietic Stem and Progenitor Cell Bioengineered Factor VIII Lentiviral Vector Gene Therapy for Hemophilia A.

Genetically modified, autologous hematopoietic stem and progenitor cells (HSPCs) represent a new class of genetic medicine. Following this therapeutic paradigm, we are developing a product candidate, designated CD68-ET3-LV CD34+, for the treatment of the severe bleeding disorder, hemophilia A. The product consists of autologous CD34+ cells transduced with a human immunodeficiency virus 1-based, monocyte lineage-restricted, self-inactivating lentiviral vector (LV), termed CD68-ET3-LV, encoding a bioengineered coagulation factor VIII (fVIII) transgene, termed ET3, designed for enhanced expression. This vector was shown capable of high-titer manufacture under clinical scale and Good Manufacturing Practice. Biochemical and immunogenicity testing of recombinant ET3, as well as safety and efficacy testing of CD68-ET3-LV HSPCs, were utilized to demonstrate overall safety and efficacy in murine models. In the first model, administration of CD68-ET3-LV-transduced stem-cell antigen-1+ cells to hemophilia A mice resulted in sustained plasma fVIII production and hemostatic correction without signs of toxicity. Patient-derived, autologous mobilized peripheral blood (mPB) CD34+ cells are the clinical target cells for ex vivo transduction using CD68-ET3-LV, and the resulting genetically modified cells represent the investigational drug candidate. In the second model, CD68-ET3-LV gene transfer into mPB CD34+ cells isolated from normal human donors was utilized to obtain in vitro and in vivo pharmacology, pharmacokinetic, and toxicology assessment. CD68-ET3-LV demonstrated reproducible and efficient gene transfer into mPB CD34+ cells, with vector copy numbers in the range of 1 copy per diploid genome equivalent without affecting clonogenic potential. Differentiation of human CD34+ cells into monocytes was associated with increased fVIII production, supporting the designed function of the CD68 promoter. To assess in vivo pharmacodynamics, CD68-ET3-LV CD34+ cell product was administered to immunodeficient mice. Treated mice displayed sustained plasma fVIII levels and no signs of product related toxicity. Collectively, the findings of the current study support the preclinical safety and efficacy of CD68-ET3-LV CD34+.

MiRNA regulation of Tip110 expression and self-renewal and differentiation of human CD34+ hematopoietic cells.

Tip110 expression regulates hematopoiesis, but the regulatory mechanisms during hematopoiesis are not fully understood. There are a number of putative microRNA (miRNA) binding sites identified within the Tip110 3′ untranslated region (3′UTR). In this study, we determined the relationship among Tip110 miRNA, Tip110 expression and self-renewal and differentiation of human CD34+ hematopoietic cells. Using a Tip110 3UTR-based reporter gene assay, 11 miRNA showed the specific activity toward the Tip110 3′UTR and down-regulated constitutive Tip110 mRNA expression. When human cord blood CD34+ cells were differentiated, Tip110 mRNA expression showed significant decreases. Concurrently, five miRNA showed significant increases, five miRNA showed significant decreases, and one miRNA remained unchanged. To further assess the roles of miRNA in Tip110 expression and self-renewal and differentiation of human CD34+ hematopoietic cells, human cord blood CD34+ cells were transduced to express the full-length Tip110 3′UTR RNA. Expression of the Tip110 3′UTR RNA led to significant increases of Tip110 mRNA, and the number of hematopoietic stem cells and progenitor cells. Taken together, these results show important roles of Tip110 miRNA in Tip110 expression control and Tip110 regulation of hematopoiesis and offer a possibility of using Tip110 miRNA or 3′UTR as a strategy to maintain human CD34+ hematopoietic cells.

Protein arginine methyltransferase 6 controls erythroid gene expression and differentiation of human CD34+ progenitor cells.

Hematopoietic differentiation is driven by transcription factors, which orchestrate a finely tuned transcriptional network. At bipotential branching points lineage decisions are made, where key transcription factors initiate cell type-specific gene expression programs. These programs are stabilized by the epigenetic activity of recruited chromatin-modifying cofactors. An example is the association of the transcription factor RUNX1 with protein arginine methyltransferase 6 (PRMT6) at the megakaryocytic/erythroid bifurcation. However, little is known about the specific influence of PRMT6 on this important branching point. Here, we show that PRMT6 inhibits erythroid gene expression during megakaryopoiesis of primary human CD34+ progenitor cells. PRMT6 is recruited to erythroid genes, such as glycophorin A. Consequently, a repressive histone modification pattern with high H3R2me2a and low H3K4me3 is established. Importantly, inhibition of PRMT6 by shRNA or small molecule inhibitors leads to upregulation of erythroid genes and promotes erythropoiesis. Our data reveal that PRMT6 plays a role in the control of erythroid/megakaryocytic differentiation and open up the possibility that manipulation of PRMT6 activity could facilitate enhanced erythropoiesis for therapeutic use.

Knockdown of HCK Reduces Cell Death and Erythroid Differentiation in Human CD34+ Hematopoietic Progenitor Cells

Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and increased risk of transformation to acute myeloid leukemia (AML). The identification of genes and cellular pathways active in leukemia cells but not in normal hematopoietic stem/progenitor cells (HSC) may help to understand the key steps in the MDS and AML pathogenesis and lead to new approaches to further enhance the treatment of both diseases, considered incurable with non-transplantation therapy. Src kinase family (SFK) is a central mediator in multiple oncogenic signaling pathways and some SFK members (Hck, Lyn, Fgr, Fyn) had previously been described as overexpressed or activated in leukemic cells. However, to this moment, the role of hematopoietic cell kinase (HCK), the unique SFK member restricted expressed in hematopoietic cells, had not been characterized in MDS and AML pathogenesis as well as in HSC. In order to better understand the HCK importance in hematopoiesis, we used lentiviral shRNA vectors to knockdown the HCK expression in primary human CD34+ HSC. The HCK levels were reduced in approximately 70-80% (shHCK) compared to the control lentiviral shRNA (shControl-GFP). To promote erythroid differentiation, human CD34+ transduced cells were grown in methylcellulose for 7 days and in liquid media for another 6 days. During this experiment, shHCK cells showed decreased cell viability (fold change compared to shControl-GFP = 0.55, P<.0001, n=3) combined with an increase in CD71+ expression (fold change compared to shControl-GFP = 3, P<.01, n=3), indicating a delay in erythroid differentiation. As expected, shControl-GFP cells showed a decreased GATA1 expression during erythroid differentiation. Meanwhile, shHCK cells did not modulate GATA1 expression. Interestingly, without any stimulus, HCK knockdown in CD34+ cells significantly decreased apoptosis (AnnexinV+ cells) compared to shControl-GFP (fold change = 0.52, P<.01, n=4). Attempts have been made to overexpress HCK in CD34+ HSC, however more than 80% cells were apoptotic and further assays were not possible. Thus, in HSC, HCK participates of erythroid differentiation and apoptosis signaling. According to the HCK importance on HSC and that SFK inhibitors are undergoing early phase clinical testing, a specific inhibitory activity compound for HCK, named iHCK-37, had been developed by Dr Maurizzio Botta. We tested this compound on primary normal human CD34+ cells originated from healthy donors bone marrow samples and also from cord blood units. The iHCK-37 treatment did not change proliferation, survival and death of these normal CD34+ cells. Conversely, MDS and AML CD34+ cells treated with the same drug exhibited a dose-dependent growth inhibition. Likewise, following iHCK-37 treatment of MDS and AML total bone marrow mononuclear cells, the BFU-E and CFUs colony numbers were significantly decreased compared to untreated cells (vehicle). We also observed a potent in vitro antiproliferative activity of iHCK-37 against a panel of leukemia cell lines, with uM IC50 values in AML (5.0 - 5.8uM) and chronic myeloid leukemia (9.1 - 19.2uM). In addition, the combinatory in vitro treatment of iHCK-37 and 5-Azacitidine (Aza) also demonstrated additive effects relative to either drug alone. Interestingly, iHCK-37 or iHCK-37 plus Aza treatments of dysplastic and leukemia cells enhanced apoptosis and resulted in increased BAX and reduced BCL-XL protein levels. This result could be clinically relevant for MDS, as Aza is the only treatment available for higher-risk MDS, but with low response rates and frequent induced resistance and refractoriness over time. In summary, we herein have shown that HCK mRNA knockdown of normal CD34+ cells resulted in growth inhibition, decreased cell death and reduced erythroid differentiation, suggesting that HCK is essential for normal hematopoiesis. We presume that the deregulation of HCK pathway in leukemic cells might be crucial for MDS and AML pathogenesis. On the other hand, the inhibition of HCK protein activity with a specific inhibitor was able to restore the apoptotic pathways of leukemic cells, acting on cancer cells without alter any signaling of normal cells. Moreover, the specific inhibitor may have antineoplastic effect that can even be additive to current available drugs. Our study adds new insights to the role of HCK in MDS and AML as well as into potential new anticancer treatment strategies. DisclosuresNo relevant conflicts of interest to declare.

In vitro transdifferentiation of human cultured CD34+ stem cells into oligodendrocyte precursors using thyroid hormones

The extent of myelination on the axon promotes transmission of impulses in the neural network, any disturbances in this process results in the neurodegenerative condition. Transplantation of oligodendrocyte precursors that supports in the regeneration of axons through myelination is an important step in the restoration of damaged neurons. Therefore, in the present study, the differentiation of human CD34+ stem cells into oligodendrocytes was carried out. The pure human CD34+ culture developed from the stem cells obtained from a peripheral blood of a donor were subjected to oligodendrocyte differentiation medium (ODM). The ODM at a concentration of 40ng/ml thyroxine, 40ng/ml 3,3′,5-tri-iodo-thyronine showed distinct morphological changes from day 6 to 9 with cells exhibiting conspicuous stellate morphology and extensive foot processes. The real-time PCR analysis showed prominent expression of Olig2, CNPase, PDGFRα and PLP1/DM20 in the differentiated cells confirming the formed cells are oligodendrocyte precursors. The expression of these genes increased from days 6 to 9 corresponding to the morphological changes observed with almost no expression of GFAP+ cells. The distinct CNPase activity was observed in these differentiated cells compared to normal CD34+ stem cells correlating with results of real-time PCR conclusively explains the development of oligodendrocytes from human CD34+ stem cells.

The DPY30 subunit in SET1/MLL complexes regulates the proliferation and differentiation of hematopoietic progenitor cells

AbstractEpigenetic mechanisms, including histone modifications, have emerged as important factors influencing cell fate determination. The functional role of H3K4 methylation, however, remains largely unclear in the maintenance and differentiation of hematopoietic stem cells (HSCs)/hematopoietic progenitor cells (HPCs). Here we show that DPY30, a shared core subunit of the SET1/MLL family methyltransferase complexes and a facilitator of their H3K4 methylation activity, is important for ex vivo proliferation and differentiation of human CD34+ HPCs. DPY30 promotes HPC proliferation by directly regulating the expression of genes critical for cell proliferation. Interestingly, while DPY30 knockdown in HPCs impaired their differentiation into the myelomonocytic lineage, it potently promoted hemoglobin production and affected the kinetics of their differentiation into the erythroid lineage. In an in vivo model, we show that morpholino-mediated dpy30 knockdown resulted in severe defects in the development of the zebrafish hematopoietic system, which could be partially rescued by coinjection of dpy30 messenger RNA. Taken together, our results establish a critical role of DPY30 in the proliferation and appropriate differentiation of hematopoietic progenitor cells and in animal hematopoiesis. Finally, we also demonstrate a crucial role of DPY30 in the growth of several MLL1-fusion–mediated leukemia cell lines.

Exposure to ionizing radiations can cause hazardous effects on differentiation of human CD34+ hematopoietic stem cells

Background Despite being a strong mutagen, causing several genetic and chromosomal aberrations, ionizing radiation in the form of gamma rays and heavy ion radiation is becoming increasingly important in medical therapies and cancer. Heavy ions especially the particle beam of carbon ion are high energy radiations that is considered to be extremely hazardous during occupational radiological emergencies, manned space missions, high altitude flights, accidents casualties etc. Healthy blood cells in human body arise by the differentiation of a very small population of pluripotent hematopoietic stem cells that have the capacity of self renewal throughout their lifespan. Effects of radiation whatsoever minimum on these unique cells thus have greater impact on the human system in both long term and short term scenario which might currently lead to an idea of a new concept leading to cancer stem cells.

Integrated analysis of miRNA and mRNA during differentiation of human CD34+ cells delineates the regulatory roles of microRNA in hematopoiesis

In the process of human hematopoiesis, precise regulation of the expression of lineage-specific gene products is critical for multiple cell-fate decisions that govern cell differentiation, proliferation, and self-renewal. Given the important role of microRNAs (miRNAs) in development and differentiation, we examined the global expression of miRNA in CD34(+) cells during lineage specific hematopoiesis and found 49 miRNAs to be differentially expressed, with functional roles in cellular growth and proliferation, and apoptosis. miR-18a was upregulated during erythropoiesis and downregulated during megakaryopoiesis. miR-145 was upregulated during granulopoiesis and down regulated during erythropoiesis. Megakaryopoitic differentiation resulted in significant alteration in the expression of many miRNAs that are believed to play critical roles in the regulation of B and T cell differentiation. Target prediction analyses on three different miRNA databases indicated that TargetScan outperformed microCosm and miRDB in identifying potential miRNA targets associated with hematopoietic differentiation process. An integrated analysis of the observed miRNAs and messenger RNAs (mRNAs) resulted in 87 highly correlated miRNA-mRNA pairs that have major functional roles in cellular growth and proliferation, hematopoietic system development, and Wnt/B-catenin and Flt 3 signaling pathways. We believe that this study will enhance our understanding on the regulatory roles of miRNA in hematopoiesis by providing a library of mRNA-miRNA networks.

Role of Nfatc2 in Proliferation and Differentiation of Human CD34+ Hematopoietic Stem Cells

Role of Nfatc2 in Proliferation and Differentiation of Human CD34+ Hematopoietic Stem Cells

Expression of Junctional Adhesion Molecule-C on the Surface of Platelets Supports Adhesion, but not Differentiation, of Human CD34+ Cells in Vitro

Background: CD34⁺ progenitor cells play an important role in haematopoiesis and vascular homeostasis. The aim of the present study was to investigate the role of platelet-derived junctional adhesion molecule-C (JAM-C) in adhesion and differentiation of human CD34⁺ cells in vitro, as well as its association with platelet-derived P-selectin in patients with coronary artery disease. Methods and Results: Using flow cytometry we observed that JAM-C expression on the surface of washed platelets is increased after activation with thrombin receptor activating peptide-6 in vitro and correlated with platelet-derived P-selectin expression in patients with coronary artery disease (r=0.326, P=0.007). The role of JAM-C and its counter receptor Mac-1 in adhesion of human CD34⁺ cells over immobilized platelets was investigated by using a neutralizing soluble protein (sJAM-C-Fc) and a monoclonal antibody against JAM-C or integrin Mac-1 (CD11b/CD18). Treatment with soluble JAM-C-Fc or anti-JAM-C or anti-Mac-1, but not with control-Fc or IgG1, resulted in a significantly decreased adhesion of human CD34⁺ cells to platelets under static conditions (P<0.05). In order to validate our findings under high shear stress we performed flow chamber experiments. In a similar manner, inhibiting JAM-C interaction with Mac-1 resulted in a significantly decreased adhesion of CD34⁺ cells over immobilized platelets under high shear stress (P<0.05). Colony forming unit assays and coculture assays revealed that inhibition of JAM-C/Mac-1 axis did not influence the platelet-mediated differentiation of CD34⁺ cells to endothelial cells or to macrophages/foam cells. Conclusions: Taken together a platelet-derived JAM-C supports CD34⁺ cell adhesion, a mechanism potentially involved in homing as well as domiciliation of human CD34⁺ cells.

Role of NFAT1 (NFATc2) in Proliferation and Differentiation of Human CD34+ Hematopoietic Stem Cells

Abstract 4833 Background:NFAT1 (NFATc2) transcription factor is known to modulate allogeneic T-cell mediated immune responses in the post transplant setting by affecting release of bioactive cytokines such as IFN- γ and IL-2 from donor CD4+T-Cells. Cyclosporine-A (CSA) inhibits NFAT1 activation via the phosphatase-Calcineurin and has been shown to be effective in prophylaxis and treatment of graft versus host disease (GVHD). However, little is known of the role of NFAT1 transcription factor and its inhibition, in affecting differentiation and proliferation of CD34+ Hematopoietic Stem Cells. Methods:Mononuclear cells (MNC) were prepared using Ficoll Paque density gradient method from human cord blood (Umbilical Cord blood Units kindly provided by Dr. Pablo Rubinstein, MD, New York Blood Center). These MNC’s were enriched for CD34+ HSC using magnetic bead separation (Auto Macs, Miltenyi). The resulting purity of HSC routinely exceeded >90%. This CD 34 + cell population was seeded at a density of 60,000 cells/ml in 12 well plates in CellGenix (serum free media) supplanted by the following cytokines: IL-3 (5ng/ml), FLT-3L (100 ng/ml), SCF (50 ng/ml), G-CSF (30 ng/ml) and GM-CSF (5ng/ml). Cells were cultured under normoxic conditions for 7 days at 37.0C with 5% CO2 either in the presence or absence of CSA at concentration 2μM. We examined cell proliferation and differentiation by the following cell surface markers- CD34/CD33/HLA-DR/CD71/ and CD14. Cell counts were enumerated (Coulter) at baseline, day 2, 5, 7 and multiplied by FACS proportions to calculate absolute numbers of specified cell populations. Experiments were conducted with and without CSA in culture media, a known inhibitor of NFAT1 dephosphorylation. Cells were harvested on Day 0, 2, 5 and 7. Results:Flow cytometry analysis of CD 34 + cells grown in absence of CSA showed progressive decline of CD34 expression over days 2, 5 and 7 and progressive increased in CD33 and HLA-DR surface expression. In the presence of CSA however, we saw a rapid rise of CD33 (41.0% versus 28.1%), HLA-DR (44.4% versus 29.1%) and CD 71 (24.4% versus 12.7%) cell fractions on Day 7, compared to culture conditions when CSA was absent., No difference was noted in total cell count in CSA treated (701,666.6 ± 7637 (Mean and SD))and untreated (643,333 ± 5166 cells) conditions in culture by Day 7. Discussion:The above results suggest that inhibition of NFAT1 signaling in CD34+ HSC using CSA results in more rapid differentiation towards the myelomonocytic and erythrocytic lineages as seen by flow cytometry. This data suggests that NFAT1 may participate in hematopoietic differentiation of CD34 progenitors in these cytokine conditions, and further that loss of NFAT1 signaling may allow enhanced kinetics of differentiation of CD34 + HSC towards myelomonocytic and erythroid lineages. Further understanding of NFAT1 regulation of normal HSC may have implications for routine CSA administration in patients undergoing allogeneic transplantation as well as potential role in myeloid malignancies resulting from NFAT1 dysregulation. Acknowledgment:Farrow Fellowship Award [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

IKAROS isoform 6 enhances BCR-ABL1-mediated proliferation of human CD34+ hematopoietic cells on stromal cells

The BCR-ABL1 induces chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). Recent studies revealed high ratios of loss of the IKZF1 gene which encodes IKAROS in BCR-ABL1+ ALL and lymphoblastic crisis (LBC) of CML. However, little is known about the cooperativity between the aberrant IKAROS and BCR-ABL1 in primary human hematopoietic cells. We investigated the effects of expression of BCR-ABL1 and/or IK6, a natural dominant negative isoform of IKAROS, on proliferation and differentiation of human CD34+ cord blood cells with or without human bone marrow-derived stromal cells which support early B cell differentiation. Cell proliferation was remarkably enhanced by co-expression of BCR-ABL1 and IK6, with reduced expression of glycophorin A and increased expression of CD41, especially on stromal cells, compared with expression of BCR-ABL1 alone that resulted in expansion of erythroid progenitors. Interestingly, p190BCR-ABL1 showed higher dependency on stromal cells to stimulate cell growth with IK6, than p210BCR-ABL1. Furthermore, the cooperation was found to depend on direct cell adhesive interaction of hematopoietic progenitors with stromal cells. These findings suggest that IK6 and BCR-ABL1 synergistically contribute to leukemogenesis in human bone marrow stromal microenvironment, and may provide a clue to elucidate the mechanisms of leukemogenesis of Ph+ ALL and CML-LBC.

A critical role for SHP2 in STAT5 activation and growth factor–mediated proliferation, survival, and differentiation of human CD34+ cells

AbstractSHP2, a cytoplasmic protein-tyrosine phosphatase encoded by the PTPN11 gene, plays a critical role in developmental hematopoiesis in the mouse, and gain-of-function mutations of SHP2 are associated with hematopoietic malignancies. However, the role of SHP2 in adult hematopoiesis has not been addressed in previous studies. In addition, the role of SHP2 in human hematopoiesis has not been described. These questions are of considerable importance given the interest in development of SHP2 inhibitors for cancer treatment. We used shRNA-mediated inhibition of SHP2 expression to investigate the function of SHP2 in growth factor (GF) signaling in normal human CD34+ cells. SHP2 knockdown resulted in markedly reduced proliferation and survival of cells cultured with GF, and reduced colony-forming cell growth. Cells expressing gain-of-function SHP2 mutations demonstrated increased dependency on SHP2 expression for survival compared with cells expressing wild-type SHP2. SHP2 knockdown was associated with significantly reduced myeloid and erythroid differentiation with retention of CD34+ progenitors with enhanced proliferative capacity. Inhibition of SHP2 expression initially enhanced and later inhibited STAT5 phosphorylation and reduced expression of the antiapoptotic genes MCL1 and BCLXL. These results indicate an important role for SHP2 in STAT5 activation and GF-mediated proliferation, survival, and differentiation of human progenitor cells.

Tight Control of STAT5 Activity Determines Human CD34-Derived Interstitial Dendritic Cell and Langerhans Cell Development

Despite the crucial function of dendritic cells (DC) in immunity, the molecular mechanisms regulating human DC development remain poorly defined. STAT5 regulates various hematopoietic lineages and is activated by GM-CSF, a critical cytokine in DC development. In this study, we investigated the role of STAT5 during differentiation of human CD34(+) hematopoietic progenitors into precursor DC (pre-DC) and their subsequent differentiation toward interstitial DC and Langerhans cells. Inhibiting STAT5 activity by dominant-negative STAT5 promoted Langerhans cell commitment of hematopoietic progenitors but resulted in loss of pre-interstitial DC development, showing subset-specific regulation. Increasing the low endogenous STAT5 activity by ectopic STAT5 activation downregulated expression of the critical DC transcription factor PU.1 and abrogated commitment to either DC lineage. In contrast, high STAT5 activity was beneficial in already committed pre-DC: terminal DC differentiation was associated with increased endogenous STAT5 phosphorylation levels, JAK2-STAT5 inhibition reduced terminal DC differentiation, and conditional STAT5 activation in pre-DC improved development of BDCA-1(+), DC-SIGN(+), and Langerin(+) DC with normal maturation and T cell stimulation. These data show that STAT5 critically regulates human DC development, with specific requirements for the level of STAT5 activation at distinct differentiation stages. By regulating STAT5 activity, cytokines present at specific locations and under different pathophysiological conditions can determine the fate of DC precursors.

Abstract 1950: Suppression of RISC-independent decoy and RISC-mediated RNA-pairing activities of microRNA-328 is required for maturation-arrest and enhanced survival of blast crisis CML progenitors

Abstract MicroRNAs (miRs) and heterogeneous ribonucleoproteins (hnRNPs) are post-transcriptional gene regulators that bind to mRNA in a sequence-specific manner. We showed that hnRNP-E2 inhibits myeloid maturation of bone marrow (BM) progenitors from chronic myelogenous leukemia patients in myeloid blast crisis (CML-BC) by suppressing CEBPA mRNA translation. We report here that loss of miR-328 is induced by BCR/ABL and specifically occurs in CML-BC, and its restored expression rescues differentiation and impairs clonogenic potential of BCR/ABL+ BM progenitors. Accordingly, miR-328 increases during granulocytic differentiation of human CD34+ and mouse LSK BM stem/progenitor cells. Mechanistically, BCR/ABL uses the MAPK-hnRNP-E2 pathway to suppress C/EBPα and miR-328 expression as pharmacologic inhibition of and/or shRNAs against these molecules efficiently restore miR-328 expression. Interestingly, two functional C/EBPα binding sites are present in the miR-328 promoter and positively regulate its transcription. We also show that maturation of differentiation-arrested BCR/ABL+ blasts requires direct interaction of hnRNP-E2 with the miR-328 C-rich regions. Moreover, imatinib treatment restores miR-328 expression, thus allowing its direct binding to hnRNP E2 independent from the RISC complex. Importantly, physiological miR-328 expression decreased hnRNP E2 binding to the uORF/spacer region of endogenous CEBPA mRNA (decoy activity). This, in turn, releases CEBPA mRNA from hnRNP E2 translation inhibition and allows in vitro and in vivo BCR/ABL+ cell differentiation. Although hnRNP E2 was not found in complex with the basic RISC components in BCR/ABL+ cells, miR-328 was found associated to Dicer and Ago2, suggesting that miR-328 also acts through base-pairing with the 3′UTR of mRNA targets in a RISC-dependent manner. In fact, miR-328 suppresses PIM1 protein but not mRNA expression and this effect requires the integrity of the PIM1 3′UTR. Indeed, forced expression of a wild type, but not a kinase-deficient, PIM1 lacking the 3′UTR into miR-328-expressing cells fully rescues BCR/ABL clonogenicity, suggesting that miR-328-induced inhibition of PIM1 accounts for reduced survival of miR-328-infected CML-BCCD34+ blasts. To demonstrate that miR-328 acts on PIM1 in a RISC-dependent manner, we mutated the miR-328 in the seed sequence (miR-328-Mut) while retaining its C-rich character. As expected, miR-328-Mut interacted with hnRNP-E2 and rescued C/EBPα-mediated differentiation, but did not silence PIM1 expression. Thus, the discovery of dual activities for miR-328 which affect myeloid differentiation and survival not only adds a layer to the complexity of mechanisms regulating CML-BC but also highlights the ability of miRNAs to alter mRNA metabolism by acting as molecular decoys for RNA binding proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1950.

Junctional Adhesion Molecule A Expressed on Human CD34 + Cells Promotes Adhesion on Vascular Wall and Differentiation Into Endothelial Progenitor Cells

To investigate the role of junctional adhesion molecule A (JAM-A) on adhesion and differentiation of human CD34(+) cells into endothelial progenitor cells. Tissue healing and vascular regeneration is a multistep process requiring firm adhesion of circulating progenitor cells to the vascular wall and their further differentiation into endothelial cells. The role of JAM-A in platelet-mediated adhesion of progenitor cells was investigated by adhesion assays in vitro and with the help of intravital fluorescence microscopy in mice. Preincubation of human CD34(+) progenitor cells with soluble JAM-A-Fc (sJAM-A-Fc) resulted in significantly decreased adhesion over immobilized platelets or inflammatory endothelium under high shear stress in vitro and after carotid ligation in vivo or ischemia/reperfusion injury in the microcirculation of mice. Human CD34(+) cells express JAM-A, as defined by flow cytometry and Western blot analysis. JAM-A mediates differentiation of CD34(+) cells to endothelial progenitor cells and facilitates CD34(+) cell-induced reendothelialization in vitro. Pretreatment of human CD34(+) cells with sJAM-A-Fc resulted in increased neointima formation 3 weeks after endothelial denudation in the carotid arteries of nonobese diabetic/severe combined immunodeficient mice. These results indicate that the expression of JAM-A on CD34(+) cells mediates adhesion to the vascular wall after injury and differentiation into endothelial progenitor cells, a mechanism potentially involved in vascular regeneration. Human CD34(+) cells express JAM-A, mediating their interaction with platelets and endothelial cells. Specifically, JAM-A expressed on human CD34(+) progenitor cells regulates their adhesion over immobilized platelets or inflammatory endothelium under high shear stress in vitro and after carotid ligation in vivo or ischemia/reperfusion injury in the microcirculation of mice. Moreover, it mediates differentiation of CD34(+) cells to endothelial progenitor cells and facilitates reendothelialization.