Potential Of Hematopoietic Progenitors Research Articles

Divergent expression of Neurl3 from hemogenic endothelial cells to hematopoietic stem progenitor cells during development

Prior to the generation of hematopoietic stem cells (HSCs) from the hemogenic endothelial cells (HECs) mainly in the dorsal aorta in midgestational mouse embryos, multiple hematopoietic progenitors including erythro-myeloid progenitors and lymphoid progenitors are generated from yolk sac HECs. These HSC-independent hematopoietic progenitors have recently been identified as major contributors to functional blood cell production until birth. However, little is known about yolk sac HECs. Here, combining integrative analyses of multiple single-cell RNA-sequencing datasets and functional assays, we reveal that Neurl3-EGFP, in addition to marking the continuum throughout the ontogeny of HSCs from HECs, can also serve as a single enrichment marker for yolk sac HECs. Moreover, while yolk sac HECs have much weaker arterial characteristics than either arterial endothelial cells in the yolk sac or HECs within the embryo proper, the lymphoid potential of yolk sac HECs is largely confined to the arterial-biased subpopulation featured by the Unc5b expression. Interestingly, the B lymphoid potential of hematopoietic progenitors, but not for myeloid potentials, is exclusively detected in Neurl3-negative subpopulations in midgestational embryos. Taken together, these findings enhance our understanding of blood birth from yolk sac HECs and provide theoretical basis and candidate reporters for monitoring step-wise hematopoietic differentiation.

Evidence of Increased Hemangioblastic and Early Hematopoietic Potential in Chronic Myeloid Leukemia (CML)-derived Induced Pluripotent Stem Cells (iPSC)

Hemangioblasts derived from mesodermal lineage are the earliest precursors of hematopoietic stem cells and endothelial cells. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are the only experimental systems in which these cells can be assayed and quantified. We show here using CML-derived iPSC and blast-cell colony forming (Bl-CFC) assays that hemangioblasts are highly expanded in CML derived iPSC as compared to human H1-ESC-derived hemangioblasts. BCR-ABL signaling pathway is intact in these cells with evidence of CRK-L phosphorylation which is reduced by the use of Imatinib. Hematopoietic progenitor assays generated using blast-CFC demonstrates also a highly increased hematopoietic progenitor potential of these cells as compared to H1-ESC. The same results were also obtained using hematopoietic progenitor assays via embryoid body formation. In CML iPSC, we have also found a significant reduction of Aryl Hydrocarbon Receptor (AHR) expression. Further inhibition of AHR using StemRegenin (SR1), an AHR antagonist, led to an increase of blast-cell colonies in CML iPSC whereas the use of an AHR agonist inhibited blast cell colonies. Thus, our results show for the first time, the possibility of establishment of a myeloproliferative phenotype using patient-derived iPSC and the presence of a major expansion of the hemangioblast compartment and hemangioblast-derived hematopoietic progenitors in this context. They also suggest that the AHR signaling pathway could represent a novel druggable target in CML.

The SIX1 homeobox Gene Is a Novel Therapeutic Target in CALM-AF10 Leukemogenesis

Background : The CALM-AF10 translocation is found 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we hypothesized that identification of non-HOXA CALM-AF10 effector genes could potentially yield novel therapeutic targets. To discover novel CALM-AF10-regulated genes, we took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal within CALM. Using microarrays, we identified a set of genes that showed decreased expression in response to the CRM1 inhibitor, Leptomycin B (LMB), similar to Hoxa genes, in murine CALM-AF10 leukemia cells. Then using RNA-sequencing, we discovered a set of genes increased in murine hematopoietic stem cells transduced with CALM-AF10. There were 11 genes that were both decreased in response to LMB and increased in response to CALM-AF10, which included the Hoxa gene cluster, as well as Six1. Similar to HOXA genes, SIX1 is a homeobox gene that is associated with embryogenesis and is quiescent post-embryologically. Additionally, SIX1 and its cofactor EYA2 have been found to be overexpressed in numerous solid tumors, and inhibitor of the SIX1/EYA2 complex has recently been described. While there is evidence of a role for SIX1 in solid tumors, its role in leukemias has not been explored. Objective: To evaluate the role of SIX1 in CALM-AF10 leukemias. Design/Methods: RT-qPCR and Chromatin Immunoprecipitation (ChIP) were performed using bone marrow progenitors transduced with CALM-AF10 or an empty vector, with and without LMB. Methylcellulose colony assays assessed the ability of SIX1 to enhance self-renewal of hematopoietic progenitors. An inhibitor of the Six1/Eya2 interaction (compound 8430) was used to evaluate cell proliferation. Downstream targets of Six1 were evaluated using RT-qPCR in CALM-AF10 cells treated with Six1/Eya2 inhibitor (8430). Results: RT-qPCR confirmed overexpression of SIX1 in CALM-AF10 leukemia cells, and showed decreased SIX1 expression in the presence of LMB. Furthermore, ChIP revealed that CALM-AF10 binds to the SIX1 gene locus. Overexpression of SIX1 in fetal liver progenitors was sufficient to increase self-renewal potential. The 8430 Six1/Eya2 inhibitor slowed cell growth in CALM-AF10 cells compared to cells treated with DMSO alone. Finally, downstream targets such as Slc2a1, Cdk2, and Cyclina2 were decreased in 8430-treated CALM-AF10 leukemia cells. Conclusions: The SIX1 homeobox gene is highly expressed during embryogenesis, and its expression is silenced post-embryogenesis. Through an initial unbiased screen, we discovered that Six1 may play a role in CALM-AF10 leukemogenesis. We have determined that Six1 expression is upregulated in the presence of CALM-AF10. Further, we have shown a potential oncogenic role for Six1, as it was able to increase the self-renewal potential of hematopoietic progenitors. The role of Six1 in CALM-AF10 leukemia is further supported by the ability of a SIX1/EYA2 inhibitor to slow the growth of CALM-AF10 leukemia cells and decrease the expression of downstream targets of SIX1. These observations suggest that Six1 plays a pathogenic role in leukemogenesis, and may be a novel therapeutic target in CALM-AF10 leukemias. Disclosures No relevant conflicts of interest to declare.

Inducible Forward Programming of Human Pluripotent Stem Cells to Hemato-endothelial Progenitor Cells with Hematopoietic Progenitor Potential.

(Stem Cell Reports 14, 122–137; January 14, 2020) The authors wish to apologize for errors in the Supplemental Experimental Procedures. The units of the antibiotic concentrations were accidentally misrepresented as being in ng and g instead of μg. The units have now been corrected as follows: “Long term iPSC cultivation was performed as ML in CM in the presence of puromycin (0.3 μg mL−1) and/or zeocin (0.5 μg mL−1) (both InvivoGen, Toulouse, France) to prevent vector-silenced SLGE-iPSC throughout the cultivation.” “Puromycin (0.3 μg mL−1) and zeocin (0.5 μg mL−1) (both InvivoGen) were applied 24 h post-transduction.” These corrections do not influence the validity of the results or conclusions of this paper. Inducible Forward Programming of Human Pluripotent Stem Cells to Hemato-endothelial Progenitor Cells with Hematopoietic Progenitor PotentialLange et al.Stem Cell ReportsDecember 12, 2019In BriefLange, Schambach, and colleagues established an inducible hemato-endothelial forward programming protocol, utilizing the combinatorial and timely regulated overexpression of hematopoietic master regulators SCL, LMO2, GATA2, and ETV2 (SLGE). Regulated induction of this combination in SLGE-iPSCs generated large numbers of hemato-endothelial progenitor cells (CD144+/CD73–) capable of producing hematopoietic progenitor cells (CD45+/CD34+) with multi-lineage potential. Furthermore, transcriptome analysis identified potential regulators of definitive hematopoiesis. Full-Text PDF Open Access

Inducible Forward Programming of Human Pluripotent Stem Cells to Hemato-endothelial Progenitor Cells with Hematopoietic Progenitor Potential.

SummaryInduced pluripotent stem cells (iPSCs) offer a promising platform to model early embryonic developmental processes, to create disease models that can be evaluated by drug screens as well as proof-of-concept experiments for regenerative medicine. However, generation of iPSC-derived hemato-endothelial and hematopoietic progenitor cells for these applications is challenging due to variable and limited cell numbers, which necessitates enormous up-scaling or development of demanding protocols. Here, we unravel the function of key transcriptional regulators SCL, LMO2, GATA2, and ETV2 (SLGE) on early hemato-endothelial specification and establish a fully inducible and stepwise hemato-endothelial forward programming system based on SLGE-regulated overexpression. Regulated induction of SLGE in stable SLGE-iPSC lines drives very efficient generation of large numbers of hemato-endothelial progenitor cells (CD144+/CD73–), which produce hematopoietic progenitor cells (CD45+/CD34+/CD38–/CD45RA−/CD90+/CD49f+) through a gradual process of endothelial-to-hematopoietic transition (EHT).

SIX1 Is a Novel Effector Gene in CALM-AF10 Leukemia

Background: The CALM-AF10 translocation is detected in ~10% of T-cell acute lymphoblastic leukemias (T-ALLs), and in some acute myeloid leukemias (AMLs). CALM-AF10 leukemias are characterized by high expression of proleukemic HOXA genes, which serve a critical role in hematopoiesis. We hypothesized that identification of novel CALM-AF10 effector genes may yield new therapeutic targets in this difficult to treat leukemia. We took advantage of our prior observation that the nuclear export factor CRM1/XPO1 tethers CALM-AF10 to HOXA genes by interacting with a nuclear export signal (NES) in CALM. Using next generation sequencing, we determined that, SIX1, similar to HOXA genes, is increased in CALM-AF10 leukemias and decreased in response to CRM1 inhibition with Leptomycin B (LMB). Design/Methods: RT-qPCR and Chromatin Immunoprecipitation were performed using both bone marrow progenitors and murine embryonic fibroblasts (MEFs) transduced with CALM-AF10 or an empty vector, with and without LMB. The ability of SIX1 to enhance self-renewal of hematopoietic progenitors was examined by measuring the colony-forming ability of transduced fetal liver hematopoietic progenitor cells. CRISPR-Cas9 was used to silence SIX1 in Human Embryonic Kidney 293 (HEK293) cells. Results: RT-qPCR confirmed overexpression of SIX1 in both CALM-AF10 transduced MEFs and CALM-AF10 leukemias, with decreased SIX1 expression observed in the presence of LMB. ChIP analysis showed that CALM-AF10 binds to the SIX1 gene locus. Overexpression of SIX1 in fetal liver cells was sufficient to increase the self-renewal potential of colony-forming progenitors. SIX1 was successfully knocked out in HEK293 cells without a significant effect on HEK293 proliferation. Conclusions: The SIX1 homeobox gene is highly expressed during development and its expression is silenced post-embryogenesis. Increased SIX1 expression has been reported in numerous solid tumors. We have determined that SIX1 is upregulated in CALM-AF10 leukemias, and increases the self-renewal potential of hematopoietic progenitors. Using CRISPR-Cas9 to silence SIX1, we have demonstrated that SIX1 is not essential for cell survival, and that its inhibition may impair CALM-AF10 leukemia cell proliferation. Thus, SIX1 may play a pathogenic role in leukemogenesis and is a potential therapeutic target in CALM-AF10 leukemias. Disclosures No relevant conflicts of interest to declare.

Gene editing of PKLR gene in human hematopoietic progenitors through 5' and 3' UTR modified TALEN mRNA.

Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD.

Effects of Delta-Like Noncanonical Notch Ligand 1 Expression of Human Fetal Liver Hepatoblasts on Hematopoietic Progenitors.

Although the hepatic and hematopoietic progenitors of the liver are well characterized, the interactions between these two lineages remain mostly elusive. Hepatoblasts express delta-like noncanonical Notch ligand 1 (Dlk1), whose cleaved extracellular domain can become a soluble protein. We assessed the effects of DLK1 gene expression knockdown in cultures of total fetal liver cells. Furthermore, we separated Dlk1+ hepatoblasts from the total liver cell fraction and investigated effects of direct cell contact. Dlk1− cells were cultured either without Dlk1+ hepatoblasts, in direct contact with hepatoblasts, or separated from hepatoblasts by a porous membrane in inserts to inhibit cell contact but allow free exchange of molecules. Expression of the hepatic and hematopoietic genes, colony forming unit potential of various hematopoietic progenitors, and cell numbers and types were investigated. We found that DLK1 knockdown in total fetal liver cell cultures decreased total cell numbers. The expression of hepatic progenitor genes and mature hematopoietic genes was affected. Hematopoietic BFU-E and CFU-GM colony numbers were reduced significantly. The depletion of Dlk1+ hepatoblasts in culture decreased the potential of all hematopoietic progenitors to form colonies of all types and reduced the percentage of mature hematopoietic cells. The addition of hepatoblasts in inserts to Dlk1− cells further decreased the potential to form the CFU-GM and CFU-GEMM colonies and the percentage of mature hematopoietic cells but increased total cell numbers. Conclusively, direct contact of Dlk1 supports hematopoietic progenitor expansion and functionality that cannot be reconstituted in coculture without direct cell contact.

DYNAMIC CHANGES IN THE BONE MARROW CELLULAR COMPOSITION OF RATS IN THE BURN WOUNDS HEALING IN NORM AND IN CONDITIONS OF HYPERGLYCEMIA

Relevance. Today the study of the potential of hematopoietic progenitors is particular relevance in connection with the use of hematopoietic cells as the main material of transplantation for the treatment of various diseases including trophic ulcers in diabetes mellitus.
 Objective is study the dynamic fluctuations of the cellular composition of the bone marrow (ВM) of rats according to myelogram indices during the healing of a burn wound in normal conditions and in conditions of hyperglycemia.
 Materials and methods. The study of dynamics (3, 7, 14, 21 days) of burn wound of skin of rats of the Vistar line without somatic pathology "control group" (n=24) and rats (n=24) with experimental streptotrozine diabetes mellitus (CD) (Blood glucose level – 24,24±0,79 mmol/L against 8,03±0,4 mmol/L in the control group). Under etheric anesthesia the burn was simulated using copper plates in the form of an ellipse. The total area of skin burn was 18-20 % of the body surface. The study of myelogram was carried out according to the standard method of M. Arinkkin the material of the bone marrow was washed from the distal end of the femur. The stroke was fixed with ethanol and stained with Romanovsky-Gimza. On each glass 500 cell elements were counted the number of cells of each species was determined and converted to percentages. Microscopy was performed under imesium at an increase of 100×10. All indicators in healing dynamics were compared with those of myelogram which were determined in the group (n = 12) of healthy rats who did not reproduce the burn. Their indicators were considered to be referential values (RZ). Statistical data was processed using IBM SPSS Statistics 23.
 Results. In normal wound healing is accompanied by a general increase in the proliferation of myeloid splenectomy with a slight oppression of 7 days. At the same time in all terms of observation the proliferation of elements of erythroid origin of BM decreases but their functional capacity and hemoglobin saturation remains normal. The differentiation of cellular elements in the BM also increases but differ in terms. Up to 3 days mature granulocytes and monocytes are actively differentiated. At 7th day in the BM the relative number of eosinophils and basophils is increased. Probably such a redistribution reflects the regulation of cell reproduction in the BM for their release into the peripheral bloodstream and subsequent participation in the process of regeneration of the connective tissue which provides normal physiological healing.
 Under conditions of hyperglycemia the difference in the cellular composition of the BM is already at the stage of the common polypotent precursors: the proliferation of common cells of lymphoid origin increases 1,5-fold and due to this the content of myeloid contraction is reduced. On all terms of observation in 1,4 times the proliferation of erythrocyte precursors predominates and the total proliferative activity of granulocytic precursors increases by 2,5 times. Due to this in 1,5-2 times the differentiation of granulocytes decreases. Particularly sensitive to hyperglycemia was the growth of basophils and monocytes which in BM was 1,5-2,8 times smaller and the dynamics of changes in terms of observation also differed from control.
 Conclusion: The effect of hyperglycemia on proliferation capacity and activity differentiation in BM causes a redistribution of cellular elements reflected on the number and functional ability of cells involved in providing and regulating signaling during connective tissue regeneration. Changes in the relative content of cellular progenitors in BM and the late release of cells from BM to peripheral blood can cause a violation of the connective tissue regeneration scenario and cause trophic ulcers or non-healing wounds in diabetes.

Abstract 1978: SON and its splice variants regulate MLL1/2 complex-mediated H3K4me3 and transcription of leukemia-associated genes

Abstract SON is a poorly characterized nuclear protein that is particularly abundant in hematopoietic cells/organs and embryonic stem cells. This protein was recently identified as a splicing co-factor required for proper cell cycle progression and maintenance of stem cell pluripotency. Although SON's function in RNA splicing was recently highlighted, SON was originally identified as a DNA-binding protein and a potential regulator of transcription. However, the mechanism by SON controls transcription and its disease relevance are completely unknown. To investigate SON's function in genome-wide gene regulation, we performed chromatin immunoprecipitation-sequencing (ChIP-seq) in K562 human leukemia cells using SON antibodies. SON ChIP-seq results demonstrated that most of SON-binding sites are located within the promoter of target genes which include signaling mediators, transcription factors and cell cycle regulators. Our ChIP-qPCR revealed that knockdown of SON causes enhanced recruitment of the mixed lineage leukemia (MLL)1/2 complexes, but not MLL3/4 and SET1A/B complexes, to the SON target chromatin, resulting in significant increase of tri-methylation of histone H3 lysine 4 (H3K4me3) levels. Surprisingly, the C-terminus of SON directly interacts with MLL-binding region of menin and interrupts menin interaction with MLL1/2. These findings demonstrate an inhibitory effect of menin-SON interaction on menin-MLL1/2 interaction, reducing H3K4me3 and MLL1/2 complex assembly. In addition to full-length SON (SON F), two C-terminally truncated splice variants of SON (SON B and E) have been predicted in genome databases. To address the clinical significance of SON splice variants, we examined whether SON splice variants are differentially expressed in the condition of acute myeloid leukemia (AML). Interestingly, the expression levels of alternatively spliced SON isoforms, but not full-length SON, were significantly increased in human AML patient bone marrow/blood samples and mouse models of leukemia. The short isoforms of SON retain its DNA-binding ability, thereby competing with the full-length SON for target chromatin interaction. However, the short isoforms lack the menin-binding ability and could not inhibit MLL1/2 complex assembly. Importantly, overexpression of a short isoform of SON increased MLL complex-mediated H3K4me3 and markedly enhanced replating potential of hematopoietic progenitors. Taken together, our study reveals that the MLL1/2 complex activity is competitively regulated by full-length SON and its alternatively spliced isoforms, and that target genes of MLL1/2-menin are aberrantly controlled by overexpressed “short SON” in AML patients. Furthermore, our findings strongly suggest the significant roles of SON splice variants in aberrant transcriptional initiation in leukemia and leukemic stem cell maintenance. Citation Format: Jung-Hyun Kim, Eun Young Park, Joshua K. Stone, Thomas W. Butler, Steve Lim, Eun-Young Erin Ahn. SON and its splice variants regulate MLL1/2 complex-mediated H3K4me3 and transcription of leukemia-associated genes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1978.

SON and Its Alternatively Spliced Isoforms Control MLL Complex-Mediated H3K4me3 and Transcription of Leukemia-Associated Genes

Dysregulation of MLL complex-mediated histone methylation plays a pivotal role in gene expression associated with diseases, but little is known about cellular factors modulating MLL complex activity. Here, we report that SON, previously known as an RNA splicing factor, controls MLL complex-mediated transcriptional initiation. SON binds to DNA near transcription start sites, interacts with menin, and inhibits MLL complex assembly, resulting in decreased H3K4me3 and transcriptional repression. Importantly, alternatively spliced short isoforms of SON are markedly upregulated in acute myeloid leukemia. The short isoforms compete with full-length SON for chromatin occupancy but lack the menin-binding ability, thereby antagonizing full-length SON function in transcriptional repression while not impairing full-length SON-mediated RNA splicing. Furthermore, overexpression of a short isoform of SON enhances replating potential of hematopoietic progenitors. Our findings define SON as a fine-tuner of the MLL-menin interaction and reveal short SON overexpression as a marker indicating aberrant transcriptional initiation in leukemia.

Cdk4 and Cdk6 cooperate in counteracting the INK4 family of inhibitors during murine leukemogenesis

AbstractCdk4 and Cdk6 are related protein kinases that bind d-type cyclins and regulate cell-cycle progression. Cdk4/6 inhibitors are currently being used in advanced clinical trials and show great promise against many types of tumors. Cdk4 and Cdk6 are inhibited by INK4 proteins, which exert tumor-suppressing functions. To test the significance of this inhibitory mechanism, we generated knock-in mice that express a Cdk6 mutant (Cdk6 R31C) insensitive to INK4-mediated inhibition. Cdk6R/R mice display altered development of the hematopoietic system without enhanced tumor susceptibility, either in the presence or absence of p53. Unexpectedly, Cdk6 R31C impairs the potential of hematopoietic progenitors to repopulate upon adoptive transfer or after 5-fluorouracil–induced damage. The defects are overcome by eliminating sensitivity of cells to INK4 inhibitors by introducing the INK4-insensitive Cdk4 R24C allele, and INK4-resistant mice are more susceptible to hematopoietic and endocrine tumors. In BCR-ABL–transformed hematopoietic cells, Cdk6 R31C causes increased binding of p16INK4a to wild-type Cdk4, whereas cells harboring Cdk4 R24C and Cdk6 R31C are fully insensitive to INK4 inhibitors, resulting in accelerated disease onset. Our observations reveal that Cdk4 and Cdk6 cooperate in hematopoietic tumor development and suggest a role for Cdk6 in sequestering INK4 proteins away from Cdk4.

In vivo evidence for an instructive role of fms-like tyrosine kinase-3 (FLT3) ligand in hematopoietic development

Cytokines are essential regulators of hematopoiesis, acting in an instructive or permissive way. Fms-like tyrosine kinase 3 ligand (FLT3L) is an important cytokine for the development of several hematopoietic populations. Its receptor (FLT3) is expressed on both myeloid and lymphoid progenitors and deletion of either the receptor or its ligand leads to defective developmental potential of hematopoietic progenitors. In vivo administration of FLT3L promotes expansion of progenitors with combined myeloid and lymphoid potential. To investigate further the role of this cytokine in hematopoietic development, we generated transgenic mice expressing high levels of human FLT3L. These transgenic mice displayed a dramatic expansion of dendritic and myeloid cells, leading to splenomegaly and blood leukocytosis. Bone marrow myeloid and lymphoid progenitors were significantly increased in numbers but retained their developmental potential. Furthermore, the transgenic mice developed anemia together with a reduction in platelet numbers. FLT3L was shown to rapidly reduce the earliest erythroid progenitors when injected into wild-type mice, indicating a direct negative role of the cytokine on erythropoiesis. We conclude that FLT3L acts on multipotent progenitors in an instructive way, inducing their development into myeloid/lymphoid lineages while suppressing their megakaryocyte/erythrocyte potential.

RUNX1c Expression Identifies an Enriched Subpopulation of Hematopoietic Progenitor Cells Derived From Human Pluripotent Stem Cells.

Abstract 2294The ability of human embryonic stem cells (hESCs) to differentiate into any cell lineage makes them an important resource for studies of developmental biology. hESC-derived hematopoietic stem cells (HSCs) developed through in vitro culture systems could provide an unlimited supply of material for replacement of defective blood lineages in vivo. However, to date, there has been only limited ability to isolate functional HSCs from hESCs. Runx1 is a key hematopoietic transcription factor vital to the development of the early embryo. Previous studies have demonstrated the onset of Runx1c to correlate directly with the emergence of definitive HSCs in the murine embryo. Similarly, expression of RUNX1c increases during differentiation of hESCs to hematopoietic cells. This particular feature makes RUNX1c an especially attractive tool for tracking the development of potential HSCs from hESCs. Therefore, we have created a novel reporter system in hESCs wherein both 1 kb of the basal promoter as well as the +24 intronic enhancer of RUNX1c drive expression of the tdTomato fluorochrome. Transgenic hESC lines stably expressing tdTomato from RUNX1c regulatory elements were generated using the Sleeping Beauty transposon system. Successfully engineered clones were selected based on constitutive expression of a GFPzeocin fusion protein which was also included in the transgene. To induce differentiation, the spin-embryoid body (EB) method was used with fully defined media. Differentiating cells were analyzed for tdTomato expression. Sorted tdTomato+ cells were evaluated for RUNX1c expression and hematopoietic stem/progenitor cell potential (CFC assay). Undifferentiated RUNX1c reporter hESC clones were uniformly tdTomato-negative. These clones are shown to express the tdTomato transgene concurrent with the expression of endogenous RUNX1c, as analyzed by flow cytometry and qRT-PCR. Expression was first seen at day 12 with a peak between days 14–16 of differentiation. Cells sorted for expression of tdTomato showed a significant increase of endogenous RUNX1c expression compared to tdTomato-negative cells. Using fluorescence microscopy, we were able to detect tdTomato+ cells with hematopoietic morphology budding from the EBs after 12–15 days. Together, these results validate the accuracy of this reporter system. tdTomato+ cells were found to be enriched for hematopoietic progenitor potential, as demonstrated by increased ability to form hematopoietic colonies in methylcelluose (430 +/− 143 per 105 cells) as compared to both tdTomato negative (131 +/− 15 per 105 cells) and unsorted cells (163 +/− 28 per 105 cells). Interestingly and unexpectedly, flow cytometric analysis demonstrated that only 5–10% of CD34+CD45+ cells expressed tdTomato. The first CD45+ population was found to lack tdTomato expression. Likewise, the first emerging tdTomato+ cells did not express CD45. Of the extracellular markers examined in this study, CD31 and CD43 were found to be expressed on all tdTomato+ cells, while CD31+ and CD43+ populations, though initially tdTomato−, were found to gradually increase in tdTomato expression to 80% and 90% by day 20, respectively. Emerging CD34+ cells lack tdTomato expression with an increase to around 10% of cells, plateauing at day 15. While CD45+ cells initially lack tdTomato expression, they gain expression over time, until days 19–22, when 50–90% of CD45+ cells co-express tdTomato (RUNX1c). Therefore, this hESC RUNX1c reporter system appears to accurately define timing of RUNX1c expression. The finding that emerging RUNX1c+ cells have increased hematopoietic progenitor cell potential mimics what has been found in mice. This, combined with our finding that only a fraction of CD34+CD45+ cells express Runx1c, suggests that one reason hESC-derived hematopoietic cells fail to engraft in immunodeficient mice is that only a small percent of differentiated cells that express hematopoietic surface antigens actually have hematopoietic stem/progenitor cell activity. Current studies focus on more in-depth analysis of tdTomato expressing cells using both single-cell qRT-PCR as well as LTC-IC and engraftment assays to test for functionality. Furthermore, we are currently testing an iPSC line stably expressing the same RUNX1c:tdTomato transgene to test how its RUNX1c expression in iPSCs compares to hESCs during hematopoietic differentiation. Disclosures:No relevant conflicts of interest to declare.

Parathyroid hormone regulates the distribution and osteoclastogenic potential of hematopoietic progenitors in the bone marrow

Parathyroid hormone (PTH) increases both the number of osteoclast in bone and the number of early hematopoietic stem cells (HSCs) in bone marrow. We previously characterized the phenotype of multiple populations of bone marrow cells with in vitro osteoclastogenic potential in mice. Here we examined whether intermittent administration of PTH influences these osteoclast progenitor (OCP) populations. C57BL/6 mice were treated with daily injections of bPTH(1-34) (80 µg/kg/day) for 7 or 14 days. We found that PTH caused a significant increase in the percentage of TN/CD115(+) CD117(high) and TN/CD115(+) CD117(int) cells (p < .05) in bone marrow on day 7. In contrast, PTH decreased the absolute number of TN/CD115(+) CD117(low) cells by 39% on day 7 (p < .05). On day 14, there was no effect of PTH on osteoclast progenitor distribution in vivo. However, PTH treatment for 7 and 14 days did increase receptor activator of NF-κB ligand (RANKL)- and macrophage colony-stimulating factor (M-CSF)-stimulated in vitro osteoclastogenesis and bone resorption in TN/CD115(+) cells. In the periphery, 14 days of treatment increased the percentage and absolute numbers of HSCs (Lin(-) CD117(+) Sca-1(+) ) in the spleen (p < .05). These data correlated with an increase in the percent and absolute numbers of HSCs in bone marrow on day 14 (p < .05). Interestingly, the effects on hematopoietic progenitors do not depend on osteoclast resorption activity. These results suggest that in vivo PTH treatment increased in vitro osteoclastogenesis and resorption without altering the number of osteoclast precursors. This implies that in vivo PTH induces sustained changes, possibly through an epigenetic mechanism, in the in vitro responsiveness of the cells to M-CSF and RANKL.

Reduced EBF expression underlies loss of B‐cell potential of hematopoietic progenitors with age

Aging is accompanied by a reduction in the generation of B lymphocytes leading to impaired immune responses. In this study, we have investigated whether the decline in B lymphopoiesis is due to age-related defects in the hematopoietic stem cell compartment. The ability of hematopoietic stem cells from old mice to generate B cells, as measured in vitro, is decreased 2-5-fold, while myeloid potential remains unchanged. This age-related decrease in B-cell potential is more marked in common lymphoid progenitors (CLP) and was associated with reduced expression of the B-lineage specifying factors, EBF and Pax5. Notably, retrovirus-mediated expression of EBF complemented the age-related loss of B-cell potential in CLP isolated from old mice. Furthermore, transduction of CLP from old mice with a constitutively active form of STAT5 restored both EBF and Pax5 expression and increased B-cell potential. These results are consistent with a mechanism, whereby reduced expression of EBF with age decreases the frequency with which multipotent hematopoietic progenitors commit to a B-cell fate, without altering their potential to generate myeloid cells.

Wnt and Notch signaling pathways selectively regulating hematopoiesis

Hematopoietic stem and progenitor cells (HSPCs) are the source of all blood cells in the adult body. The pool of HSPCs is formed during embryogenesis process through a well-characterized succession of intra-embryonic regions and organs. The spatial and temporal restrictions in definitive hematopoietic development and the signaling molecules involved are of great interest as these may prove useful for generating and expanding these clinically important cell populations ex vivo. To elucidate the mechanism by which definitive HSPCs expand during this limited developmental time frame, we analyzed the spatial and temporal programmed gene expression patterns of Wnt and Notch signaling members during hematopoietic development. Genes related to the Wnt signaling pathway were up-regulated in E10.5 aorta-gonad-mesonephros (AGM) and E14.5 fetal liver corresponding to the inherent proliferation potential of hematopoietic progenitors, whereas genes related to the Notch signaling pathway were identified as up-regulated in E10.5 AGM, and bone marrow coincides with the maintenance of undifferentiation state of hematopoietic progenitors. Our findings suggest that Wnt and Notch signalings are integrated and are selectively regulating hematopoiesis. The spatial and temporal balance between Wnt and Notch signaling orchestrates the precise progression of hematopoietic progenitors.

Bone marrow engraftment but limited expansion of hematopoietic cells from multipotent germline stem cells derived from neonatal mouse testis

Multipotent germline stem (mGS) cells derived from neonatal mouse testis, similar to embryonic stem (ES) cells, differentiate into various types of somatic cells in vitro and produce teratomas after inoculation into mice. In the present work, we examined mGS cells for hematopoietic progenitor potential in vitro and in vivo. mGS cells were differentiated on OP9 stromal cells and induced into Flk1(+) cells. Flk1(+) cells were sorted and replated on OP9 stromal cells with various cytokines and emerging hematopoietic cells were analyzed for lineage marker expression by fluorescein-activated cell sorting, progenitor activity by colony assay, and stem cell transplantation assay. mGS cells, like ES cells, produce hematopoietic progenitors, including both primitive and definitive erythromyeloid, megakaryocyte, and B- and T-cell lineages via Flk1(+) progenitors. When transplanted into the bone marrow (BM) of nonobese diabetic/severe combined immunodeficient (NOD/SCID) gammac(null) mice directly, mGS-derived green fluorescent protein (GFP)-positive cells were detected 4 months later in the BM and spleen. GFP(+) donor cells were also identified in the Hoechst33342 side population, a feature of hematopoietic stem cells. However, these mGS-derived hematopoietic cells did not proliferate in vivo, even after exposure to hematopoietic stressors, such as 5-fluorouracil (5FU) injection or serial transplantation. mGS cells produced multipotent hematopoietic progenitor cells with myeloid and lymphoid lineage potential in vitro and localized in the BM after intra-BM injection but, like ES cells, failed to expand or show stem cell repopulating ability in vivo.

VEGF and IHH rescue definitive hematopoiesis in Gata-4 and Gata-6–deficient murine embryoid bodies

Murine embryonic stem cells can be differentiated into embryoid bodies (EBs), which serve as an in vitro model recapitulating many aspects of embryonic yolk sac hematopoiesis. Differentiation of embryonic stem cells deficient in either Gata-4 or Gata-6 results in EBs with disrupted visceral endoderm (VE). While lack of VE has detrimental effects on hematopoiesis in vivo, it is unclear whether lack of VE affects hematopoiesis in EBs. Therefore, we compared Gata-4 null (G4N) and Gata-6 null (G6N) EBs with wild-type EBs to assess their ability to commit to hematopoietic cells. EB VE formation was examined using cell-sorting techniques and analysis visceral endoderm gene expression. Hematopoietic progenitor potential of EBs cultured under various conditions was assessed using colony-forming assays. Definitive erythroid, granulocyte-macrophage, and mixed colonies were significantly reduced in G4N and G6N EBs compared to wild-type EBs. Vascular endothelial growth factor (VEGF) expression and secretion were also reduced in both G4N and G6N EBs, consistent with VE serving as a site of VEGF production. Addition of exogenous VEGF(165), to EB cultures completely rescued definitive colony-forming cells in G4N and G6N EBs. This rescue response could be blocked by addition of soluble Flk-1 Fc to EB cultures. Similarly, addition of exogenous Indian hedgehog to EB cultures also recovers the diminishment in definitive hematopoiesis in a reversible manner. These results suggest that the absence of VE in G4N and G6N EBs does not prevent emergence of definitive progenitors from EBs. However, the decreased level of VEGF and Indian hedgehog production in VE devoid G4N and G6N EBs attenuates definitive hematopoietic progenitor cell expansion.

Human and Simian Immunodeficiency Viruses De-Regulate Early Hematopoiesis through a Novel Nef/PPAR-γ/STAT5 Signaling Pathway

Infection of primates by HIV-1 and SIV induces multiple hematological abnormalities of central hematopoietic origin. Although these defects greatly contribute to the pathophysiology of HIV-1 infection, the molecular basis for altered BM function remains unknown. Here we show that when cynomolgus macaques were infected with SIV, the multipotent potential of their hematopoietic progenitor cells was lost, and this correlated with down regulation of STAT5A and STAT5B expression. However, forced expression of STAT5B entirely rescued the multipotent potential of the hematopoietic progenitor cells. In addition, an accessory viral protein required for efficient SIV and HIV replication and pathogenicity, “Negative factor” (Nef), was essential for <SIV-mediated impairment of the multipotent potential of hematopoietic progenitors ex vivo and in vivo. This newly uncovered property of Nef was both conserved between HIV-1 and SIV strains and entirely dependent upon the presence of PPARγ in targeted cells. Further, PPARγ agonists mimicked Nef activity by inhibiting STAT5A and STAT5B expression and hampering the functionality of hematopoietic progenitors both ex vivo and in vivo. These findings have extended the role of Nef in the pathogenicity of HIV-1 and SIV and reveal a pivotal role for the PPARγ/STAT5 pathway in the regulation of early hematopoiesis. This study may provide a basis for investigating the potential therapeutic benefits of PPARγ antagonists in both patients with AIDS and individuals with hematopoietic disorders.