Mouse Multipotent Progenitor Research Articles

The PRC2 Co-Factor JARID2 Regulates Hematopoiesis through Non-Canonical Mechanisms

Epigenetic mechanisms play an important role in the determination of hematopoietic stem and progenitor cell fate through the modification of chromatin architecture. Jarid2 is a co-factor of the polycomb repressive complex 2 (PRC2) that recruits this complex to different sites in the genome to catalyze the repressive epigenetic mark of Histone 3 Lysine 27 tri-methylation (H3K27me3). Previous studies from our lab have demonstrated that Jarid2 functions as a tumor suppressor in chronic myeloid neoplasms by recruiting PCR2 to silence self-renewal gene expression programs in committed hematopoietic progenitor cells. In the absence of Jarid2, mouse multipotent progenitor (MPP) cells gain ectopic self-renewal in normal hematopoiesis, and neoplastic transformation is accelerated from JAK2V617F-mutant MPN. However, our previous studies showed that in Jarid2-null MPPs, only about half of the upregulated genes were associated with a loss of H3K27me3, suggesting that Jarid2 might have important non-canonical (PCR2-independent) functions in hematopoiesis, which forms the scientific premise of the current study. When bound to PRC2, the lysine 116 (K116) residue of Jarid2 becomes methylated by the catalytic subunit Ezh2. Jarid2K116 methylation was shown to be indispensable for the regulation of the PRC2 catalytic activity through competing with the H3K27me3 binding to PRC2. Here, we created a separation of function mouse model to study the non-canonical functions of Jarid2 in hematopoiesis. Through CRISPR/Cas9 gene targeting, we created a genetic knock in mouse model by mutating lysine 116 to alanine (K116A), a point mutation that cannot be methylated by PRC2. We generated a Vav-cre:Jarid2fl/K116A(K116A) mouse model, that express one K116A mutated copy of Jarid2, while the other floxed copy will be conditionally knocked out by Vav-cre recombinase system in hematopoietic stem and progenitor cells (HSPCs). We hypothesized if Jarid2K116A HSPCs functionally phenocopy Vav-Cre:Jarid2fl/fl (Jarid2-KO) HSPCs then the function of Jarid2 in these cells is largely PCR2-dependent. However, if the output of Jarid2-K116A HSPCs resembles that of Vav-Cre:Jarid2fl/+ (Jarid2-HET) HSPCs, then this suggests Jarid2 has previously undescribed non-canonical functions in hematopoiesis. To characterize the functional effects of this mutation, we performed competitive transplantation of stem and progenitor subsets. Transplantation of MPP1 (Lineage-/c-Kit+/Sca1+/CD135-/CD48-/CD150-) cells revealed that Jarid2-K116A MPPs behaved similarly to Jarid2-KO MPPs. Recipients of both Jarid2-K116A and Jarid2-KO MPPs displayed higher peripheral blood (53% and 60%, respectively) and bone marrow (33% and 43%, respectively) engraftment compared to the control (12% and 3%, respectively) and Jarid2-HET (37.5% and 11%, respectively) MPPs. Additionally, Jarid2-K116A mutation enhanced donor MPP1 chimerism over recipient cells and promoted MPP1 self-renewal, similarly to what we previously described for Jarid2-KO MPP1 cells. These results suggest that Jarid2 function in MPP1 cells is largely dependent on PRC2. However, competitive transplantation of Jarid2-K116A HSCs (HSCs; Lineage-/c-Kit+/Sca1+/CD135-/CD48-/CD150+) revealed a striking phenotype. Jarid2-K116A HSCs showed a neomorphic phenotype compared to control, Jarid2-HET and Jarid2-KO HSCs in both primary and secondary transplantations. Jarid2-K116A mutation significantly enhanced the peripheral blood and bone marrow myeloid engraftment (both 40% increase). Furthermore, Jarid2-K116A enhanced HSC fitness and self-renewal with these mutant HSCs able to gain increased chimerism in the HSC pool and increased donor-derived HSC number in the bone marrow of recipient mice (Figure 1). These results suggest that the unmethylated version of Jarid2-K116 exerts a neomorphic effect on HSC function confirming the importance of the post-translational modification at the K116 residue of Jarid2 in hematopoiesis. Thus far, our studies suggest that Jarid2 function is dependent on PRC2 in MPPs, but may act independently of PRC2 in HSCs. Ongoing studies are focused on examining the importance of PCR2-related Jarid2 function in malignant hematopoiesis, and determining if the ratio of methylated to unmethylated JARID2 influences leukemic transformation of human chronic myeloid disorders. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mouse multipotent progenitor 5 cells are located at the interphase between hematopoietic stem and progenitor cells

Hematopoietic stem cells (HSCs) and distinct multipotent progenitor (MPP) populations (MPP1-4) contained within the Lin−Sca-1+c-Kit+ (LSK) compartment have previously been identified using diverse surface-marker panels. Here, we phenotypically define and functionally characterize MPP5 (LSK CD34+CD135−CD48−CD150−). Upon transplantation, MPP5 supports initial emergency myelopoiesis followed by stable contribution to the lymphoid lineage. MPP5, capable of generating MPP1-4 but not HSCs, represents a dynamic and versatile component of the MPP network. To characterize all hematopoietic stem and progenitor cells, we performed RNA-sequencing (RNA-seq) analysis to identify specific transcriptomic landscapes of HSCs and MPP1-5. This was complemented by single-cell RNA-seq analysis of LSK cells to establish the differentiation trajectories from HSCs to MPP1-5. In agreement with functional reconstitution activity, MPP5 is located immediately downstream of HSCs but upstream of the more committed MPP2-4. This study provides a comprehensive analysis of the LSK compartment, focusing on the functional and molecular characteristics of the newly defined MPP5 subset.

Comparative Single Cell Transcriptomic Analysis of the Hematopoietic System between Human and Mouse

Single cell RNA-seq has been extensively applied to the hematopoiesis of human and mouse, but the cross-species comparison still has not been thoroughly studied. Here, using the 10X single cell platform and a canonical correlation analysis (CCA, https://satijalab.org/seurat/) computational strategy, we conducted comparative transcriptomic analysis of the hematopoietic hierarchy between human and mouse. We found that the hematopoietic stem and progenitor cell (HSPC) compartments in the two species were composed of subpopulations characterized by the same set of homologous genes, and the hematopoietic lineages and transcriptional profiling in hematopoiesis were well conserved between human and mouse, indicating evolutionary similarity in the organization of hematopoietic systems. A total of 32,805 cells derived from human bone marrow CD3-CD14-CD19-CD34+ and lineage-CD117+ cell from bone marrow of C57BL/6 mice were used to construct a single-cell resolution transcriptomic atlas of HSPCs of human and mouse. We retained only orthologous genes of human and mouse collected in InParanoid (http://inparanoid.sbc.su.se). Based on known marker genes, we grouped human cells as hematopoietic stem cell (HSC), multilymphoid progenitor (MLP), granulocyte-monocyte progenitor (GMP), Pro-B cell (ProB), earliest thymic progenitor (ETP), and megakaryocytic-erythroid progenitor (MEP); and mouse cells as long-term HSCs (LTHSC), lymphoid multipotent progenitors (LMPP), multipotent progenitor (MPP), GMP, MEP, and common myeloid progenitors (CMP). tSNE plots showed that cells were preferentially clustered by species, rather than cell type, due to species specificity and batch effects (Fig A, mouse and human cells were profiled at different time). After alignment with CCA, the cells of mouse and human were well mixed and separated by same cell type categories (Fig B-C). The cells were grouped into 17 subpopulations by computational analysis (Fig C). The cluster specific genes were species conserved and share same functional themes. To obtain a detailed view on the cellular evolution from mouse to human in the HSPC system, we used mouse and human orthologous genes, and calculated an average of expression of cells in each population of human and mouse. A hierarchical cluster dendrogram indicated that cell types were highly conserved between human and mouse (Fig D). For example, MEP and GMP of mouse and human shared a very similar transcriptome pattern. Human HSC was firstly clustered with mouse LTHSC and then with mouse MPP. Further, MPP and LMPP in mouse had similar transcriptomes, which was observed in human already. We used monocle to examine the differentiation trajectory of hematopoiesis in human and mouse, and we defined the HSC and LTHSC as roots so that they located at starting points of the differentiation hierarchy. On graphic representation after application of Monocle, for both mouse and human cells three distinct branches emerged from HSC and LTHSC: Erythroid/Megakaryocytic, Myeloid, and Lymphoid (Fig E-F). We also examined levels of gene expression. As examples, for both mouse and human, Gata1 and Cd79a expression increased along the Erythroid branch and Lymphoid branch, respectively, while Procr expression decreased with differentiation. To understand the species conservation of cell populations of hematopoiesis in human and mouse, scmap was utilized to project human dataset on mouse and inferring cellular identity with the cell types defined in mouse. Most human MEP cells (85%) were mapped to mouse MEP cell types based on transcriptional similarity, suggesting likely functional similarity and species conservation. Further, 45% and 24% human HSC cells were mapped to mouse LTHSC and MPP cell types, respectively, indicating similarity of MPP and HSC (Fig G-H). Our analysis confirms evolutionary conservation between mouse and human hematopoietic systems. Figure Disclosures No relevant conflicts of interest to declare.

A Short Pulse of Prostaglandin E2 (PGE2) Induces Long Term Chromatin Changes in Hematopoietic Stem Cells Leading to Increased Self-Renewal and Engraftment

▪Hematopoietic stem cells (HSCs) offer promising treatment options for many blood diseases. We have previously identified Prostaglandin E2 (PGE2), a small molecule that increased HSC numbers in the zebrafish embryo. In an adult mammalian transplantation setting a two hour treatment significantly enhanced HSC engraftment. Currently PGE2 is being tested in a phase 2 clinical trial to improve cord blood transplants. To better understand PGE2 effect on HSCs mouse multipotent progenitors (MPP), short term (ST) HSCs, and long term (LT) HSCs were isolated via FACS and given a two hour pulse of PGE2 followed by a competitive transplantation assay. Surprisingly, PGE2 treatment mainly affected ST-HSCs by dramatically prolonging their ability to contribute to peripheral blood. The effect of the two hour treatment persisted through secondary competitive transplants in which robust peripheral blood chimerism of ST-HSCs was evident even 1.5 years after having been exposed to the drug. To elucidate underlying molecular changes gene expression right after PGE2 treatment as well as in ST-HSCs after transplantation was assessed. PGE2 target genes were divided into two categories; "transiently induced" and "permanently induced" genes. Most of the transcripts upregulated two hour after PGE2 treatment were "transiently induced" meaning that they did not continue to be differentially expressed after transplantation. In contrast, a few transcripts including chemokines such as Cxcl2, Cxcl3, members of the Fos gene family as well as Nr4a1, 2 and 3 were both upregulated right after PGE2 treatment as well as in ST-HSCs after transplantation. We classified these genes as "permanently induced". ATAC (Assay for Transposase-Accessible Chromatin)-seq analysis of the transplanted PGE2 treated cells indicated that these "permanently induced" genes maintained a distinctly open chromatin profile in both promotor and enhancer regions, whereas the "transiently induced" genes did not. Gene expression in human CD34+ cells included a signature implying CREB as the main transcription factor responsible for the acute PGE2 response. Phospho-FACS in mouse ST-HSCs and Western-blot analysis in human CD34+ cells confirmed a significant increase in CREB phosphorylation after PGE2 stimulation. Chromatin immunoprecipitation (ChIP)-seq analysis of pCREB was able to identify specific genomic regions where pCREB is recruited to after PGE2 treatment. Compared to unstimulated CD34+ cells an increased binding of pCREB could be detected in promotor regions near transcription start sites. In addition over 90% of de-novo pCREB binding occurred in intergenic and intronic regions. To determine the activation state of these putative enhancers changes in the histone mark H3K27ac and open chromatin state (via ATAC-seq) were assessed after PGE2 treatment. The data suggest that PGE2-induced pCREB binding correlates with remodeling of chromatin already after two hours of drug treatment. Furthermore chromatin sites opened by PGE2 were significantly enriched for the CREB motif both in human CD34+ cells acutely after treatment as well as in mouse ST-HSCs 1.5 years after transplant. In summary this work shows that a two hour treatment with PGE2 is sufficient to confer long-term engraftment properties to ST-HSCs. PGE triggers a chromatin remodeling event through CREB that can permanently alter epigenetic state and gene expression of ST-HSCs. Understanding the self-renewal network induced by PGE2 will not only enrich current clinical applications targeted at increasing engraftable HSC numbers but also further basic understanding of HSC self-renewal. DisclosuresZon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Comparison of Expression and Epigenetic Profiles in Human and Mouse Erythropoiesis and Megakaryopoiesis Using a Systems Biology Model

To date numerous datasets of gene expression and epigenetic profiles for mouse and human hematopoietic cells have been generated. While individual data sets for a particular cell type have been correlated, no approach exists to harness all expression and epigenetic profiles for the different types of hematopoietic cells. Our goal is to develop a systems biology platform to compare epigenetic profiles of hematopoietic cells towards a better understanding of epigenetic mechanisms governing hematopoiesis. To provide the necessary foundation to support systematic studies of hematopoiesis, we have developed the Systems Biology Repository (SBR, http://sbrblood.nhgri.nih.gov), a data "ranch" for organizing and analyzing transcriptome and epigenome data cells throughout differentiation.To populate SBR, we extracted, curated, annotated, and integrated all human and mouse hematopoietic datasets available through the Encyclopedia of DNA Elements (ENCODE), the Gene Expression Omnibus (GEO) and the Short Read Repository (SRR). These include genome-wide profiles of DNA methylation, histone methylation and acetylation, transcription factor occupancy (ChIPSeq), chromatin accessibility (DNaseISeq, ATACSeq, FAIRESeq), and coding as well as non-coding transcriptional profiles (RNASeq).To demonstrate the utility of SBR, we conducted three different analyses. The first was a vertical study of HistoneSeq (H3K4me1, H3K4me2, H3K4me3, and H3K27ac), DNA methylation and RNASeq profiles during mouse erythroid differentiation. We found a global decrease in DNA methylation from hematopoietic stem and progenitor cells (HSC) through common myeloid progenitors (CMP), erythroid progenitor cells (MEP) and erythroblasts (ERY; 92936 peaks in HSC to 14422 in ERY). The number of expressed genes (using a tags per million cutoff of 10) increased in erythroid progenitors (8901 in HSC to 10778 in CMP and 10670 in MEP) before decreasing in ERY (8654). 62% of histone marks delineating active enhancers (H3K27ac, H3K4me1) are present in both HSC and ERY, while 48% arise de novo during differentiation. In contrast, only 16% of active promoter specific histone marks (H3K4me2, H3K4me3) are present in both HSC and ERY.For a horizontal analysis we compared the DNA methylation, RNASeq, histone modification (H3K4me1, H3K4me2, H3K4me3, and H3K27ac) and transcription factor binding (GATA1 and NFE2) profiles of erythroblasts (ERY) and megakaryocytes (MEG). We found a similar relationship between gene expression and the histone and DNA methylation profiles in each cell type but differences between expression and in transcription factor occupancy. DNA methylation and H3K4me3 was enriched in the gene body of expressed genes (>36%) for both ERY (p ≤ 0.001) and MEG (p ≤ 0.01). In contrast DNA methylation was enriched in the upstream and downstream regions of non-coding RNA genes (p ≤ 0.001). Transcription factor occupancy was cell type specific: 79% of GATA1 sites are in ERY and 72% of NFE2 sites are in MEG. In erythroblasts, DNA methylation and GATA1 binding in the gene body are associated with gene silencing (4 fold difference, p ≤ 0.001), while in megakaryocytes, DNA methylation and NFE2 binding in the gene body are associated with gene activation (8 fold difference, p ≤ 0.001).We used the Mouse Genome Informatics homology map data to perform a cross-species comparison of the expression profiles of mouse and human multipotent progenitors (MPP), proerythroblasts and orthochromatic erythroblasts. We found a total of 5247 genes expressed at significantly different levels (p ≤ 0.001) between human and mouse MPP, while only 2010 genes were expressed at significantly similar levels (p ≤ 0.001). At the proerythroblast and orthochromatic erythroblast stages 7696 genes and 6571 genes were expressed at significantly different levels (p ≤ 0.001) between human and mouse respectively, while 2024 and 2560 genes were expressed at significantly similar levels (p ≤ 0.001). These data are consistent with previous studies showing differences in the transcriptional profiles of mouse and human hematopoietic cells.In summary, SBR provides a foundation to model the genetic and epigenetic landscape in both the mouse and human hematopoietic system, and enables functional correlations to be made between the species. As SBR is expanded to include data from patient cells, it will be possible to model epigenetic changes associated with disease. DisclosuresNo relevant conflicts of interest to declare.

Cross-talk between Notch and Hedgehog regulates hepatic stellate cell fate in mice

Liver repair involves phenotypic changes in hepatic stellate cells (HSCs) and reactivation of morphogenic signaling pathways that modulate epithelial-to-mesenchymal/mesenchymal-to-epithelial transitions, such as Notch and Hedgehog (Hh). Hh stimulates HSCs to become myofibroblasts (MFs). Recent lineage tracing studies in adult mice with injured livers showed that some MFs became multipotent progenitors to regenerate hepatocytes, cholangiocytes, and HSCs. We studied primary HSC cultures and two different animal models of fibrosis to evaluate the hypothesis that activating the Notch pathway in HSCs stimulates them to become (and remain) MFs through a mechanism that involves an epithelial-to-mesenchymal-like transition and requires cross-talk with the canonical Hh pathway. We found that when cultured HSCs transitioned into MFs, they activated Hh signaling, underwent an epithelial-to-mesenchymal-like transition, and increased Notch signaling. Blocking Notch signaling in MFs/HSCs suppressed Hh activity and caused a mesenchymal-to-epithelial-like transition. Inhibiting the Hh pathway suppressed Notch signaling and also induced a mesenchymal-to-epithelial-like transition. Manipulating Hh and Notch signaling in a mouse multipotent progenitor cell line evoked similar responses. In mice, liver injury increased Notch activity in MFs and Hh-responsive MF progeny (i.e., HSCs and ductular cells). Conditionally disrupting Hh signaling in MFs of bile-duct-ligated mice inhibited Notch signaling and blocked accumulation of both MF and ductular cells. The Notch and Hedgehog pathways interact to control the fate of key cell types involved in adult liver repair by modulating epithelial-to-mesenchymal-like/mesenchymal-to-epithelial-like transitions.

γ-IFN May Act As a Positive Regulator to Hematogenesis Through PI3K/Akt and JAK2/STAT5 Pathways

Abstract 4785It is known to us all that γ-IFN is a growth inhibitory to hematogenesis and plays a critical role in the pathophysiology of aplastic anemia(AA). However, γ-IFN actually is a bilateral regulatory factor. In certain experimental situations, it can facilitate the proliferation and growth of hematopoitic stem cells. Interferon regulatory factor-1 (IRF-1) and IRF-2 are the key regulators of IFN system and IRF-1 has been shown to participate in development of CD8+ T cells, Th1 differentiation, and NK cell development. Thus, IRF-1 may has crucial functions against hematopoietic stem and/or progenitors cells. To investigate the function of IRF-1and the molecular mechanism of proliferative effect of γ-IFN, IRF-1-siRNA was designed to knocked out IRF-1 gene in 32D cells, mouse multipotent progenitor cells, and the apoptosis was assayed by flow cytometry 48 hr after incubation with γ-IFN in three different concentrations. It showed that after IRF-1 gene was knocked out, γ-IFN reversed its action on hematogenesis and apoptosis,it potentiated the proliferation and growth of 32D cells. Interestingly, at this situations, the inhibitory effect of γ-IFN on Akt activation was abolished completely and reversed to enhanced Akt phosphorylation significantly. The expression of pStat5 also changed when γ-IFN only exert proliferative effect.Nevertheless,The negative effect of γ-IFN on Erk1/2 activation was not blocked after IRF-1 gene was knocked out. Taken together, γ-IFN can enhance or antagonize cell proliferation depending on the function of IRF-1 and may act as a positive regulator to hematogenesis through PI3K/Akt and JAK2/STAT5 signaling pathways. Our findings may contribute to understanding the decreased number of stem cells characteristic of aplastic anemia and will provide novel therapeutic strategies for this disease. Disclosures:No relevant conflicts of interest to declare.

Interferon-γ attenuates the survival activity of G-CSF through PI3K/Akt signaling pathway in mouse multipotent progenitor cells

Apoptosis plays an important role in the injury to stem and progenitor compartments associated with aberrant interferon-gamma (IFN-gamma) in aplastic anemia (AA), which is characterized by the loss of stem cells; however, its molecular mechanism is poorly understood. In this study, we have addressed the mechanism of the apoptotic function of IFN-gamma against hematopoietic stem and/or progenitors. Although granulocyte colony-stimulating factor (G-CSF) augmented survival and proliferative and differentiating activity in 32D cells, mouse multipotent progenitor cells, these effects were abolished by IFN-gamma and were susceptible to apoptosis with IFN-gamma. IFN-gamma attenuated Akt phosphorylated by G-CSF in a dose- and time-dependent manner. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), enhanced the inhibitory effect on Akt phosphorylated by G-CSF in collaboration with IFN-gamma, suggesting that the activity of IFN-gamma might converge on the PI3K pathway. We examined the expression of Bcl-2-associated death (Bad), which works downstream of Akt. IFN-gamma increased the Bad protein reduced by G-CSF. IFN-gamma induced apoptosis in 32D cells through the caspase pathway. Taken together, these results suggest that IFN-gamma could exert inhibitory action on stem cells and/or progenitors by interference with the PI3K/Akt signaling pathway. Our findings may contribute to understanding the decreased number of stem cells characteristic of AA.

Interferon-γ Attenuates the Survival Activity of G-CSF through PI3K/Akt Signaling Pathway in Mouse Multipotent Progenitor Cells.

Aplastic anemia (AA) is characterized by cytopenias caused by loss of stem cell and immunological relevance. We found out that impaired stem cell could be caused by excess of interferon-gamma (IFN-γ) in the serum or hypersensitivity to IFN-γ in patients with AA, notably severe AA. However, it is not clear how IFN-γ works in terms of the etiology of AA, as it exhibits diverse biological effects on cells. Thus, we investigated the essential role and the mechanism for the apoptotic function of IFN-γ against hematopoietic stem and/or progenitor cells. Although G-CSF augmented the surviving, proliferative, and differentiating activity in 32D cells, mouse multipotent progenitor cells, those effects of G-CSF were abolished by IFN-γ and they were susceptible to apoptosis with IFN-γ treatment. We focused on Akt, which was associated with the control of stem cell fate. IFN-γ did not affect the expression of Akt in 32D cells treated with G-CSF. Intriguingly, although Akt was strongly phosphorylated at Ser473 with G-CSF, IFN-γ attenuated phospho-Akt expression in dose- and time-dependent manner. As Akt is known to be phosphorylated through phosphatidylinositol-3-kinase (PI3K) pathway, we analyzed whether wortmannin, a specific inhibitor of PI3K, might have any synergistic effect on Akt phosphorylation in conjunction with IFN-γ. Wortmannin enhanced the inhibitory effect on Akt phosphorylated by G-CSF in collaboration with IFN-γ, suggesting that the activity of IFN-γ might converge on PI3K pathway. Next, to explore the downstream molecule of Akt, we examined the expression of BAD and NF-kB, which work downstream of Akt. IFN-γ increased the BAD protein reduced by G-CSF. Regardless of addition of IFN-γ, there was no alteration of NF-kB expression. IFN-γ exerted apoptosis against 32D cells through the caspase pathway. Taken together, these results suggest that IFN-γ could exert the inhibitory action on stem cells and/or progenitors by the interference of the PI3K / Akt signaling pathway in AA patients. Our findings may provide a suggestion of the treatment potential for AA such as anti-IFN-γ antibody and contribute the further understanding of AA in terms of immunological aberration.