Single-Cell Approaches to Deconvolute the Development of HSCs

Hematopoietic stem cells (HSCs) play a core role in blood development. The ability to efficiently produce HSCs from various pluripotent stem cell sources is the Holy Grail in the hematology field. However, in vitro or in vivo HSC production remains low, which may be attributable to the lack of understanding of hematopoiesis. Here, we review the recent progress in this area and introduce advanced technologies, such as single-cell RNA-seq, spatial transcriptomics, and molecular barcoding, which may help to acquire missing information about HSC generation. We finally discuss unresolved questions, the answers to which may be conducive to HSC production, providing a promising path toward HSC-based immunotherapies.

Download Full-text

Hematopoietic Stem Cells Respond Heterogeneously to Inflammatory Signals in an Age-Dependent Manner

Blood ◽

10.1182/blood.v128.22.566.566 ◽

2016 ◽

Vol 128 (22) ◽

pp. 566-566

Author(s):

Arnav Mehta ◽

Mati Mann ◽

Monika Kowalczyk ◽

Carl de Boer ◽

Jun Wang ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Immune Cell ◽

Transcriptional Regulators ◽

Rna Seq ◽

Hematopoietic Stem ◽

Aged Mice ◽

Inflammatory Signals

Abstract Hematopoietic stem cells (HSCs) have the unique responsibility to produce balanced immune cell output throughout an organism's life. Importantly, they must do so robustly despite a plethora of external stress, including frequent inflammatory challenge. With age, the accumulation of these stresses leads to impaired HSC function and myeloid-biased output. Aged HSCs are also more prone to pathological hematopoiesis, such as myeloproliferative disorder, leukemia and autoimmune diseases. However, little is known about the subcellular mechanisms that govern the inflammatory response of HSCs with age, which in turn might contribute to pathologic transformation. We show that young hematopoietic stem and progenitor cells (HSPCs) demonstrate a robust transcriptional response to toll-like receptor (TLR) ligands. Interestingly, this response is similar to that of mature immune cell types such as dendritic cells. Using single-cell proteomic assays, we found that young HSPCs secrete a diverse array of myeloid and lymphoid cytokines. However, when challenged with TLR ligands in vivo, young mice acutely increase myeloid-biased output but return rapidly to baseline hematopoietic output of both lymphoid and myeloid cells. Moreover, inflammatory challenge of young long-term HSCs in vitro did not perturb the function and output of these cells in bone marrow reconstitution experiments. In contrast to their counterparts from young mice, we found HSPCs obtained from aged mice have a diminished ability to secrete cytokines in response to TLR ligands. Furthermore, they secrete a homogenous subset of myeloid-biased cytokines. When challenged with TLR ligands in vivo, aged mice acutely increased myeloid output and maintain elevated myeloid output for several months implying memory of the inflammatory challenge. Consistent with this, we also found that pre-stimulation of aged HSCs prior to bone marrow transplant results in a sustained increase in myeloid output compared to unstimulated aged HSCs. To elucidate the differential heterogeneity between young and aged HSPCs in response to TLR signaling, we next performed single-cell RNA sequencing (RNA-seq) experiments. We found that the sustained myeloid output in aged mice after TLR stimulation is largely due to expansion of a myeloid-biased HSC subset in the aged HSC pool. By characterizing the gene expression networks that define these myeloid-biased HSCs under stimulation conditions, we were then able to identify a myeloid-biased HSC subset in both the unperturbed young and aged HSC pools. Moreover, we found that these cells are more abundant in aged mice at steady-state, and that these HSCs demonstrate a unique response to inflammatory challenge. We further identify putative transcriptional regulators, including Klf4, Klf5, Ikzf1 and Stat3, among others, that define gene expression in these myeloid-biased HSCs. We further show that loss of function of these factors can differentially alter myeloid output in young and aged mice both in vitro and in vivo. Our results demonstrate that there is a differential response of young and aged HSCs to inflammatory signals. Using single-cell RNA-seq and protein secretion studies, we elucidate the molecular heterogeneity of the HSC pool at steady state and with TLR stimulation. By resolving heterogeneous subsets of cells in both the young and aged HSC pool, and by uncovering the transcriptional regulators that influence their function, we thus propose a new model of inflammatory hematopoiesis that may have implications to understanding age-related defects in immune development. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Single cell RNA sequencing of calvarial and long bone endocortical cells

10.1101/849224 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ugur M. Ayturk ◽

Joseph P. Scollan ◽

Alexander Vesprey ◽

Christina M. Jacobsen ◽

Paola Divieti Pajevic ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cultured Cells ◽

Neutralizing Antibody ◽

Long Bone ◽

Appendicular Skeleton ◽

Rna Seq ◽

Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

Download Full-text

Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow

10.1101/2021.08.06.455158 ◽

2021 ◽

Author(s):

Zixian Liu ◽

Jinhong Wang ◽

Miner Xie ◽

Peng Wu ◽

Yao Ma ◽

...

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Pcr Analysis ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Colony Assay ◽

Megakaryocyte Progenitors ◽

Cell Colony

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in C57BL/6 mice. Single-cell transplantation and single-cell colony assay showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average eight megakaryocytes per colony) than did previously reported megakaryocyte progenitors (MkPs). Single-cell RNA-sequencing supported that these MgPs lie between HSCs and MkPs along the megakaryocyte differentiation pathway. Single-cell colony assay and single-cell RT-PCR analysis suggested the coexpression of CD41 and Pf4 is associated with megakaryocyte colony-forming activity. Single-cell colony assay of a small number of cells generated from single HSCs in culture suggested that MgPs are not direct progeny of HSCs. In this study, we propose a differentiation model in which HSCs give rise to MkPs through MgPs.

Download Full-text

Single Cell RNA-Seq Characterises Pre-Leukemic Transformation Driven By CEBPA N321D in the Hoxb8-FL Cell Line

Blood ◽

10.1182/blood-2018-99-110626 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3887-3887

Author(s):

Moosa Qureshi ◽

Fernando Calero-Nieto ◽

Iwo Kucinski ◽

Sarah Kinston ◽

George Giotopoulos ◽

...

Keyword(s):

Dendritic Cell ◽

Single Cell ◽

Cell Line ◽

Single Cells ◽

Mutant Form ◽

Rna Seq ◽

Wild Type ◽

Leukemic Transformation

Abstract The C/EBPα transcription factor plays a pivotal role in myeloid differentiation and E2F-mediated cell cycle regulation. Although CEBPA mutations are common in acute myeloid leukaemia (AML), little is known regarding pre-leukemic alterations caused by mutated CEBPA. Here, we investigated early events involved in pre-leukemic transformation driven by CEBPA N321D in the LMPP-like cell line Hoxb8-FL (Redecke et al., Nat Methods 2013), which can be maintained in vitro as a self-renewing LMPP population using Flt3L and estradiol, as well as differentiated both in vitro and in vivo into myeloid and lymphoid cell types. Hoxb8-FL cells were retrovirally transduced with Empty Vector (EV), wild-type CEBPA (CEBPA WT) or its N321D mutant form (CEBPA N321D). CEBPA WT-transduced cells showed increased expression of cd11b and SIRPα and downregulation of c-kit, suggesting that wild-type CEBPA was sufficient to promote differentiation even under LMPP growth conditions. Interestingly, we did not observe the same phenotype in CEBPA N321D-transduced cells. Upon withdrawal of estradiol, both EV and CEBPA WT-transduced cells differentiated rapidly into a conventional dendritic cell (cDC) phenotype by day 7 and died within 12 days. By contrast, CEBPA N321D-transduced cells continued to grow for in excess of 56 days, with an initial cDC phenotype but by day 30 demonstrating a plasmacytoid dendritic cell precursor phenotype. CEBPA N321D-transduced cells were morphologically distinct from EV-transduced cells. To test leukemogenic potential in vivo, we performed transplantation experiments in lethally irradiated mice. Serial monitoring of peripheral blood demonstrated that Hoxb8-FL derived cells had disappeared by 4 weeks, and did not reappear. However, at 6 months CEBPA N321D-transduced cells could still be detected in bone marrow in contrast to EV-transduced cells but without any leukemic phenotype. To identify early events involved in pre-leukemic transformation, the differentiation profiles of EV, CEBPA WT and CEBPA N321D-transduced cells were examined with single cell RNA-seq (scRNA-seq). 576 single cells were taken from 3 biological replicates at days 0 and 5 post-differentiation, and analysed using the Automated Single-Cell Analysis Pipeline (Gardeux et al., Bioinformatics 2017). Visualisation by t-SNE (Fig 1) demonstrated: (i) CEBPA WT-transduced cells formed a distinct cluster at day 0 before withdrawal of estradiol; (ii) CEBPA N321D-transduced cells separated from EV and CEBPA WT-transduced cells after 5 days of differentiation, (iii) two subpopulations could be identified within the CEBPA N321D-transduced cells at day 5, with a cluster of five CEBPA N321D-transduced single cells distributed amongst or very close to the day 0 non-differentiated cells. Differential expression analysis identified 224 genes upregulated and 633 genes downregulated specifically in the CEBPA N321D-transduced cells when compared to EV cells after 5 days of differentiation. This gene expression signature revealed that CEBPA N321D-transduced cells switched on a HSC/MEP/CMP transcriptional program and switched off a myeloid dendritic cell program. Finally, in order to further dissect the effect of the N321D mutation, the binding profile of endogenous and CEBPA N321D was compared by ChIP-seq before and after 5 days of differentiation. Integration with scRNA-seq data identified 160 genes specifically downregulated in CEBPA N321D-transduced cells which were associated with the binding of the mutant protein. This list of genes included genes previously implicated in dendritic cell differentiation (such as NOTCH2, JAK2), as well as a number of genes not previously implicated in the evolution of AML, representing potentially novel therapeutic targets. Disclosures No relevant conflicts of interest to declare.

Download Full-text

248 Immunotherapy persister cells uncovered by dynamic single-cell RNA-sequencing

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0248 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A268-A269

Author(s):

Kartik Sehgal ◽

Andrew Portell ◽

Elena Ivanova ◽

Patrick Lizotte ◽

Navin Mahadevan ◽

...

Keyword(s):

Colorectal Cancer ◽

Single Cell ◽

Rna Sequencing ◽

Ex Vivo ◽

Interferon Γ ◽

Rna Seq ◽

Persister Cells ◽

Pre Treatment

BackgroundTo understand fundamental mechanisms of immune escape, we leveraged our functional ex vivo platform of murine derived organotypic tumor spheroids (DOTS)1 to determine if drug-tolerant persister cells analogous to oncogene targeted therapies limit efficacy of programmed death (PD)-1 blockade, and to identify therapeutic vulnerabilities to overcome anti-PD-1 (αPD-1) resistance.MethodsMurine syngeneic cancer models with well-characterized response to αPD-1 therapy were chosen: MC38 (sensitive) and CT26 (partially resistant). Bulk and single-cell (sc) RNA-sequencing (RNA-seq) were performed on αPD-1 treated DOTS. In vitro culture studies were conducted with or without cytokines (100 ng/ml) or drugs (500 nM). In vivo studies in mice bearing MC38 or CT26 tumors evaluated the combinatorial strategy with PD-1 blockade. We further evaluated our findings in scRNA-seq of an αPD-1 refractory colorectal cancer (CRC) patient tumor.2ResultsBulk RNA-seq of αPD-1 treated DOTS revealed a mesenchymal resistant phenotype with upregulated TNF-α/NFκB signaling (figure 1). scRNA-seq further identified a discrete sub-population of immunotherapy persister cells (IPCs). These cells expressed a stem-like phenotype including downregulation of E2F targets indicative of quiescence, suppression of interferon-γ response genes, induction of hybrid epithelial-to-mesenchymal state, and active IL-6 signaling (figure 1). Ly6a/stem cell antigen-1 (Sca-1) and Snai1 were found to be differentially upregulated in IPCs resistant to PD-1 blockade (not shown). Sca-1 positivity was confirmed in pre-existing tumor populations in vitro (figure 2). When enriched via sorting, these cells remained more persistently Sca-1+ at 96 hours in culture of CT26 compared to MC38 cells, related to increased autocrine IL-6 production by CT26 Sca-1+ cells. Indeed, IL-6 supplementation was capable of expanding Sca-1+ cells in culture (figure 2). Sca-1+ cells expressing ovalbumin peptide were refractory to OT-1 T cell mediated killing and failed to upregulate MHC class-1 antigen presentation (H-2Kb) in response to IL-6, in contrast to interferon-γ (not shown). Analysis of RNA-seq data further identified Birc2/3 as potential targets limiting TNF-mediated apoptosis of these cells (not shown). Notably, Birc2/3 antagonism depleted Sca-1+ IPCs in vitro and significantly potentiated the impact of PD-1 blockade in vivo in MC38, and less robustly in CT26 (figure 3). Evaluation in a microsatellite-instability high CRC patient identified a pre-existent IPC subpopulation within the αPD-1 refractory pre-treatment tumor, with high SNAI1 expression compared to CRC samples in TCGA (figure 4).Abstract 248 Figure 1Bulk and single-cell (sc) RNA-sequencing (RNA-seq) of MDOTS identifies an anti-PD-1 (αPD-1) resistant subpopulation of persister cells. IgG= isotype controlAbstract 248 Figure 2Pre-existent population of stem cell antigen-1 (Sca-1)+ cells expands in response to interleukin-6 (IL-6), as characterized by flow cytometry evaluation in murine syngeneic cancer models at baseline and after purification by fluorescence-activated cell sorting (FACS). H = hoursAbstract 248 Figure 3Combination of anti-PD-1 therapy with Birc2/3 antagonism increases tumor responses and improves survival. CR = complete responseAbstract 248 Figure 4Single-cell RNA-sequencing (scRNA-seq) of a pre-treatment microsatellite-instability (MSI-H) colorectal cancer (CRC) patient tumor, refractory to anti-PD-1 (αPD-1) therapy, reveals presence of SNAI1-high immunotherapy persister cellsConclusionsHigh-resolution functional ex vivo profiling identified Sca-1+/Snai1high stem-like ‘immunotherapy persister cells‘ and uncovered their anti-apoptotic dependencies targetable with Birc2/3 antagonism to augment αPD-1 efficacy.Ethics ApprovalThis study was approved by the Dana-Farber Animal Care and Use Committee and Novartis Institutional Animal Care and Use Committee. Informed written consent to participate in Dana-Farber/Harvard Cancer Center institutional review board (IRB)-approved research protocols was obtained from the human subject. A copy of the written consent is available for review by the Editor of this journal. The study was conducted per the WMA Declaration of Helsinki and IRB-approved protocols.ReferencesJenkins RW, Aref AR, Lizotte PH, Ivanova E, Stinson S, Zhou CW, et al. Ex Vivo Profiling of PD-1 Blockade using organotypic tumor spheroids. Cancer Discov. 2018;8(2):196–668 215.Gurjao C, Liu D, Hofree M, AlDubayan SH, Wakiro I, Su MJ, et al. intrinsic resistance to immune checkpoint blockade in a mismatch repair-deficient colorectal cancer. Cancer Immunol Res 2019;7(8):1230–6.

Download Full-text

Trajectory reconstruction identifies dysregulation of perinatal maturation programs in pluripotent stem cell-derived cardiomyocytes

10.1101/2021.01.31.428969 ◽

2021 ◽

Author(s):

Suraj Kannan ◽

Matthew Miyamoto ◽

Brian L. Lin ◽

Chulan Kwon

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Transcription Factors ◽

Single Cell ◽

Pluripotent Stem Cell ◽

Directed Differentiation ◽

Trajectory Reconstruction ◽

Single Cell Rna Sequencing

ABSTRACTA primary limitation in the clinical application of pluripotent stem cell derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. In vivo, cardiomyocytes undergo numerous adaptive changes during perinatal maturation. By contrast, PSC-CMs fail to fully undergo these developmental processes, instead remaining arrested at an embryonic stage of maturation. To date, however, the precise mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, are unknown. The advent of single cell RNA-sequencing (scRNA-seq) has offered great opportunities for studying CM maturation at single cell resolution. However, perinatal cardiac scRNA-seq has been limited owing to technical difficulties in the isolation of single CMs. Here, we used our previously developed large particle fluorescence-activated cell sorting approach to generate an scRNA-seq reference of mouse in vivo CM maturation with extensive sampling of perinatal time periods. We subsequently generated isogenic embryonic stem cells and created an in vitro scRNA-seq reference of PSC-CM directed differentiation. Through trajectory reconstruction methods, we identified a perinatal maturation program in endogenous CMs that is poorly recapitulated in vitro. By comparison of our trajectories with previously published human datasets, we identified a network of nine transcription factors (TFs) whose targets are consistently dysregulated in PSC-CMs across species. Notably, we demonstrated that these TFs are only partially activated in common ex vivo approaches to engineer PSC-CM maturation. Our study represents the first direct comparison of CM maturation in vivo and in vitro at the single cell level, and can be leveraged towards improving the clinical viability of PSC-CMs.Significance StatementThere is a significant clinical need to generate mature cardiomyocytes from pluripotent stem cells. However, to date, most differentiation protocols yield phenotypically immature cardiomyocytes. The mechanisms underlying this poor maturation state are unknown. Here, we used single cell RNA-sequencing to compare cardiomyocyte maturation pathways in endogenous and pluripotent stem cell-derived cardiomyocytes. We found that in vitro, cardiomyocytes fail to undergo critical perinatal gene expression changes necessary for complete maturation. We found that key transcription factors regulating these changes are poorly expressed in vitro. Our study provides a better understanding of cardiomyocyte maturation both in vivo and in vitro, and may lead to improved approaches for engineering mature cardiomyocytes from stem cells.

Download Full-text

Single-cell RNA-seq revealed the role of Alkbh5 in reproduction

10.1101/2021.03.06.434224 ◽

2021 ◽

Author(s):

Xiaozhong Shen ◽

Gangcai Xie

Keyword(s):

Single Cell ◽

Messenger Rna ◽

Single Cells ◽

Rna Seq ◽

Single Cell Sequencing ◽

Differential Gene ◽

Higher Eukaryotes

AbstractN(6)-methyladenosine (m(6)a) is the most common internal modification of messenger RNA (mRNA) in higher eukaryotes. According to previous literature reports, alkbh5, as another demethylase in mammals, can reverse the expression of m(6)a gene in vivo and in vitro. In order to reveal the effect of Alkbh5 deletion on the level of single cells in the testis during spermatogenesis in mice, the data were compared using single-cell sequencing. In this article, we discussed the transcription profile and cell type identification of mouse testis, the expression of mitochondrial and ribosomal genes in mice, the analysis of differential gene expression, and the effects of Alkbh5 deletion, and try to explain the role and influence of Alkbh5 on reproduction at the level of single-cell sequencing.

Download Full-text

Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application

Journal of Parkinson s Disease ◽

10.3233/jpd-212934 ◽

2021 ◽

pp. 1-14

Author(s):

Satoe Hiramatsu ◽

Asuka Morizane ◽

Tetsuhiro Kikuchi ◽

Daisuke Doi ◽

Kenji Yoshida ◽

...

Keyword(s):

Stem Cell ◽

Single Cell ◽

Clinical Application ◽

Pluripotent Stem Cell ◽

3D Structure ◽

Induced Pluripotent Stem Cell ◽

Cell Aggregates ◽

Induced Pluripotent

Background: Pluripotent stem cell (PSC)-derived dopaminergic (DA) neurons are an expected source of cell therapy for Parkinson’s disease. The transplantation of cell aggregates or neurospheres, instead of a single cell suspension has several advantages, such as keeping the 3D structure of the donor cells and ease of handling. For this PSC-based therapy to become a widely available treatment, cryopreservation of the final product is critical in the manufacturing process. However, cryopreserving cell aggregates is more complicated than cryopreserving single cell suspensions. Previous studies showed poor survival of the DA neurons after the transplantation of cryopreserved fetal ventral-mesencephalic tissues. Objective: To achieve the cryopreservation of induced pluripotent stem cell (iPSC)-derived DA neurospheres toward clinical application. Methods: We cryopreserved iPSC-derived DA neurospheres in various clinically applicable cryopreservation media and freezing protocols and assessed viability and neurite extension. We evaluated the population and neuronal function of cryopreserved cells by the selected method in vitro. We also injected the cells into 6-hydroxydopamine (6-OHDA) lesioned rats, and assessed their survival, maturation and function in vivo. Results: The iPSC-derived DA neurospheres cryopreserved by Proton Freezer in the cryopreservation medium Bambanker hRM (BBK) showed favorable viability after thawing and had equivalent expression of DA-specific markers, dopamine secretion, and electrophysiological activity as fresh spheres. When transplanted into 6-OHDA-lesioned rats, the cryopreserved cells survived and differentiated into mature DA neurons, resulting in improved abnormal rotational behavior. Conclusion: These results show that the combination of BBK and Proton Freezer is suitable for the cryopreservation of iPSC-derived DA neurospheres.

Download Full-text

Regulatory Reprogramming of Erythropoiesis By DNMT3A Mutation

Blood ◽

10.1182/blood-2018-99-120110 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4343-4343

Author(s):

Janghee Woo ◽

Sandra Stehling-Sun ◽

H. Joachim Deeg ◽

Thalia Papayannopoulou ◽

Fyodor D Urnov ◽

...

Keyword(s):

Dna Methyltransferase ◽

Catalytic Domain ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Regulatory Elements ◽

Rna Seq ◽

Hematopoietic Stem ◽

Regulatory Landscapes

Abstract DNA methyltransferase 3A (DNMT3A) regulates diverse epigenetic processes, and DNMT3A mutations occur frequently in myelodysplastic syndromes (MDS), including in founding clones of MDS samples. Most DNMT3A mutations affect Arg882 (R882) in the catalytic domain of DNMT3A, and are found almost exclusively in a heterozygous state. To resolve the relationship between the genetic and epigenetic architectures of R882H+ MDS, we engineered primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) to carry heterozygous DNMT3A R882H and performed temporally resolved, genome-wide regulatory mapping via DNase-seq combined with RNA-seq during erythroid differentiation in vitro, and in an in vivo transplantation model. Compared with isogenic controls, heterozygous R882H HSPCs cells exhibited markedly impaired erythroid differentiation, accumulation of early myeloid progenitors, and diverse maturation defects. Transplantation of R882H HSPCs into W41 NSG mice revealed both impaired erythroid differentiation and preferential survival of mutant alleles in multiple hematopoietic lineages compatible with an early progenitor defect. Regulatory profiling of DNMT3A R882H heterozygous cells during differentiation via combined DNase- and RNA-seq revealed global and sequential alterations in the regulatory landscapes in mutant cells, most prominently decommissioning of thousands of regulatory regions normally found in primitive cells that mark gene loci destined for expression during later differentiation stages. Decommissioned regulatory elements in R882H heterozygotes were concentrated around genes involved in both regulation of erythropoiesis and cell-cycle control, biasing HSPC differentiation away from erythropoiesis. Similar findings were observed in CD34+-selected bone marrows from 33 patients with MDS, comparing heterozygous DNMT3A R882H and wild type. Collectively, our results indicate that DNMT3A R882H mutation reprograms early myeloid regulatory landscapes by preferentially targeting elements that control genes destined to be expressed at later stages of differentiation, resulting in a combined phenotype of impaired myeloid differentiation, impaired erythroid maturation, and preferential survival of R882H+ cells. The results provide novel mechanistic insights into the chromatin programming of erythroid differentiation and its connection with MDS. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Engineering a Hematopoietic Stem Cell Niche By Revitalizing Mesenchymal Stem Cells with Five Transcription Factors

Blood ◽

10.1182/blood-2019-125045 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5004-5004

Author(s):

Fumio Nakahara ◽

Sandra Pinho ◽

Daniel K. Borger ◽

Qiaozhi Wei ◽

Maria Maryanovich ◽

...

Keyword(s):

Stem Cells ◽

Single Cell ◽

Stromal Cells ◽

Ex Vivo ◽

Gfp Expression ◽

Hematopoietic Stem ◽

Hsc Niche ◽

Control Mscs

Hematopoietic stem cells (HSCs) are maintained by bone marrow (BM) niches in vivo, but the ability of niche cells to maintain HSCs ex vivo is markedly diminished. Expression of niche factors (Scf, Cxcl12, Vcam1 and Angpt1) by Nestin-GFP+ mesenchymal-derived stem cells (MSCs) is downregulated upon culture and lose its effect of maintaining HSC in vitro, suggesting that transcriptional rewiring may contribute to this reduced potential in cultured MSCs. To gain further insight, we searched RNA sequencing data for transcriptional regulators that were highly expressed in Nestin-GFP+ stroma, revealing 40 potential candidates. We compared the expression of these genes by real-time quantitative PCR (qPCR) in freshly isolated Nestin-GFP+ or Nestin-GFP- BM CD45-Ter119-CD31- cells, with that of cultured Nestin-GFP+ stroma. These analyses yielded 28 candidate genes after the elimination of 12 genes due to non-specific expression or lack of downregulation after culture. We cultured stromal cells isolated from Scf-GFP knock-in mice in which GFP expression reflects endogenous Scf mRNA synthesis. Upon culture, GFP expression was rapidly downregulated in these cells, demonstrating the potential of using GFP to screen for factors capable of revitalizing niche activity in cultured MSCs. We generated lentiviral vectors expressing 28 selected genes and transduced the viral mixture into cultured stromal cells derived from Scf-GFP mice. Five days after transduction, we observed re-emergence of GFP+ cells and these GFP+ cells were sorted and plated in limiting dilutions to isolate single cell-derived clones. Using this approach, we generated 16 independent GFP+ single cell-derived clones. To determine the specific combination of genes that enables cultured stromal cells to regain their capacity to maintain and expand HSCs in vitro, lineage-negative (Lin-) BM cells were co-cultured with each single cell-derived clone or control stroma. Thus, we identified 5 transcription factors (Klf7, Ostf1, Xbp1, Irf3, and Irf7; KOXII) that restored HSC niche function in cultured BM-derived MSCs. These revitalized MSCs (rMSCs) exhibited enhanced synthesis of HSC niche factors while retaining their mesenchymal differentiation capacity. In contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs in vitro showed higher repopulation capacity and enabled lethally irradiated recipient mice to survive better. Competitive reconstitution assays revealed 7-fold expansion of functional HSCs by rMSCs. Moreover, rMSCs prevented the accumulation of DNA damage in cultured HSCs, a hallmark of ageing and replication stress. To investigate the revitalization mechanism, we performed ATAC-seq in freshly sorted Scf-GFP- CD45-Ter119-CD31- cells, Scf-GFP+ CD45-Ter119-CD31- cells, rMSCs and control vector-transduced stroma. We found that revitalization of MSCs led to 9,623 peaks of open chromatin in rMSCs when compared to control MSCs. Of these, 626 open peaks were also detected in freshly isolated Scf-GFP+ cells when compared to Scf-GFP- cells. Motif analyses of the sequence at these 626 peaks revealed that myocyte enhancer factor 2c (Mef2c) was among the most significantly enriched transcription regulators. Mef2c was also expressed at high levels in both rMSCs and freshly isolated Scf-GFP+ cells compared to control cultured MSCs and freshly isolated Scf-GFP- cells by RNA-seq and real-time qPCR. To evaluate the role of Mef2c in rMSCs, we knocked down Mef2c in rMSCs by short hairpin RNA lentiviral transduction (shMef2c). We found that the expression of niche factors (Scf, Cxcl12 and Vcam1) was reduced in shMef2c-transduced compared to parental rMSCs. In addition, shMef2c transduced-rMSCs exhibited reduced (by 43%) capacity to expand HSCs in co-culture compared to shCntrl transduced-rMSCs. These results suggest a role for Mef2c as a downstream effector mediating MSC revitalization. We are now exploring the method to make these rMSCs to form new niches in vivo. Our results suggest that combination of KOXII genes are able to fully restore the niche activity in MSCs ex vivo and establish a new platform that provides critical insight in the regulatory network of the HSC niche leading to the basis toward the engineering of supportive niches for curative cell therapies. Disclosures Wei: Albert Einstein College of Medicine, Inc: Patents & Royalties. Frenette:Albert Einstein College of Medicine, Inc: Patents & Royalties; Ironwood Pharmaceuticals: Research Funding; Cygnal Therapeutics: Equity Ownership; Pfizer: Consultancy.

Download Full-text