Sequencing of RNA in single cells reveals a distinct transcriptome signature of hematopoiesis in GATA2 deficiency

Abstract Constitutional GATA2 deficiency caused by heterozygous germline GATA2 mutations has a broad spectrum of clinical phenotypes, including systemic infections, lymphedema, cytopenias, and myeloid neoplasms. Genotype–phenotype correlation is not well understood mechanistically in GATA2 deficiency. We performed whole transcriptome sequencing of single hematopoietic stem and progenitor cells from 8 patients, who had pathogenic GATA2 mutations and myelodysplasia. Mapping patients’ cells onto normal hematopoiesis, we observed deficiency in lymphoid/myeloid progenitors, also evident from highly constrained gene correlations. HSPCs of patients exhibited distinct patterns of gene expression and coexpression compared with counterparts from healthy donors. Distinct lineages showed differently altered transcriptional profiles. Stem cells in patients had dysregulated gene expression related to apoptosis, cell cycle, and quiescence; increased expression of erythroid/megakaryocytic priming genes; and decreased lymphoid priming genes. The prominent deficiency in lympho-myeloid lineages in GATA2 deficiency appeared at least partly due to the expression of aberrant gene programs in stem cells prior to lineage commitment. We computationally imputed cells with chromosomal abnormalities and determined their gene expression; DNA repair genes were downregulated in trisomy 8 cells, potentially rendering these cells vulnerable to second-hit somatic mutations and additional chromosomal abnormalities. Cells with complex cytogenetic abnormalities showed defects in genes related to multilineage differentiation and cell cycle. Single-cell RNA sequencing is powerful in resolving transcriptomes of cell subpopulations despite a paucity of cells in marrow failure. Our study discloses previously uncharacterized transcriptome signatures of stem cells and progenitors in GATA2 deficiency, providing a broad perspective of potential mechanisms by which germline mutations modulate early hematopoiesis in a human disease. This trial was registered at www.clinicaltrials.gov as NCT01905826, NCT01861106, and NCT00001620.

Download Full-text

Deregulated Gene Expression Pathways in Myelodysplastic Syndrome Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.735.735 ◽

2009 ◽

Vol 114 (22) ◽

pp. 735-735 ◽

Cited By ~ 1

Author(s):

Andrea Pellagatti ◽

Mario Cazzola ◽

Aristoteles Giagounidis ◽

Janet Perry ◽

Luca Malcovati ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Dna Damage ◽

Gene Ontology ◽

Hematopoietic Stem Cells ◽

Molecular Pathogenesis ◽

Beta Catenin ◽

Trisomy 8 ◽

Hematopoietic Stem

Abstract Abstract 735 The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies that are characterized by ineffective hematopoiesis resulting in peripheral cytopenias and a hypercellular bone marrow. In order to gain insight into the molecular pathogenesis of the MDS, we have determined the transcriptome of the hematopoietic stem cells (HSC) of 183 MDS patients and 17 healthy controls. The CD34+ cells obtained from MDS patients and healthy individuals were analyzed using Affymetrix U133 Plus2.0 arrays. Global pathway analysis using the Ingenuity software and the DAVID database has identified critical deregulated gene pathways and gene ontology (functional) groups perturbed in MDS HSC compared with normal HSC. The most significantly deregulated pathways in MDS include interferon signaling, thrombopoietin signaling and the Wnt pathway. Moreover, we have identified multiple pathways that are deregulated in specific MDS karyotypic groups and between early (subtype RA) and advanced MDS (subtype RAEB2). Among the most significantly deregulated gene pathways and ontology groups in early MDS are immunodeficiency, apoptosis and chemokine signaling, whereas advanced MDS is characterized by deregulation of the cell cycle, DNA damage response and checkpoint pathways. The clinical behavior of patients with del(5q), +8 or–7/del(7q) is different and we have identified distinct gene expression profiles and deregulated gene pathways for MDS defined by these major karyotypic groups. The most significantly deregulated gene pathways in del(5q) MDS include primary immunodeficiency signaling, Wnt/beta-catenin signaling, integrin signaling, cell cycle regulation and Huntington's disease signaling. Patients with the 5q- syndrome also show deregulation of the p53 pathway. Moreover, chromatin assembly and translation are among the most significant gene ontology groups in del(5q) MDS. We have found that MDS with the–7/del(7q) is characterized by deregulation of multiple pathways involved in cell survival, differentiation, apoptosis and growth, and include SAPK/JNK, NF-kB, PI3K/AKT and ceramide signaling pathways. Strikingly, all of the most significantly deregulated gene pathways in trisomy 8 MDS in our study concern or are associated with the immune response, and include B-cell receptor signaling, antigen presentation and CTLA4 signaling in Cytotoxic T lymphocytes pathways. These data are consistent with an immune system role in the pathogenesis of MDS with trisomy 8. Importantly, much of the deregulated pathway data generated in this study is in accord with the known biology of MDS. On the basis of our observations, we suggest a model for MDS in which immune deregulation and activation of apoptosis pathways in early MDS cells, consistent with clinically observed ineffective hematopoiesis, functions as a barrier to prevent leukemic transformation. Disruption of the DNA damage check points in advanced MDS results in an increase in the error rate of DNA repair with a concomitant increase in genomic instability, leading to evolution to AML. This is the first study to determine deregulated gene pathways and ontology groups in the HSC compartment of a large group of patients with MDS. The deregulated pathways identified are likely to be critical to the MDS HSC phenotype, provide important new insights into the molecular pathogenesis of this disorder, and may represent new targets for therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Single-Cell RNA Sequencing Reveals a Distinct Transcriptome Signature of Hematopoiesis in GATA2 Deficiency

Blood ◽

10.1182/blood-2019-124774 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3735-3735

Author(s):

Zhijie Wu ◽

Shouguo Gao ◽

Sachiko Kajigaya ◽

Jinguo Chen ◽

Rongye Shi ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Single Cell ◽

Chromosomal Abnormalities ◽

Dna Repair Genes ◽

Trisomy 8 ◽

Single Cell Rna Sequencing ◽

Gata2 Deficiency ◽

Upregulated Genes ◽

Repair Genes

Constitutional GATA2 deficiency caused by heterozygous germline GATA2 mutations has a broad spectrum of clinical phenotypes including systemic infections, lymphedema, cytopenias, myelodysplasia, and a high risk of developing myeloid leukemias. GATA2 deficiency is recognized as a major MDS predisposition syndrome, germline GATA2 mutations are found in 7% of primary MDS cases in children and adolescents. Monosomy 7 and trisomy 8 are frequent chromosomal abnormalities in GATA2 deficiency and features of disease prognosis and malignant transformation. GATA2 mutations mostly affect two zinc finger domains and broadly classify into three major categories: missense, null, and regulatory region mutations. Profound monocytopenia, B-cell and NK-cell lymphopenias, and dendritic cell deficiency are specific for GATA2 deficiency. Absence of multi-lymphoid progenitor and decreased granulocyte -macrophage progenitors occur early in disease. There are limited studies of gene expression profiles in GATA2 deficiency and these usually in bulk populations, partly due to the paucity of cells in patients. How GATA2 deficiency negatively affects hematopoiesis, especially the preferential loss of specific lineages, is poorly understood. We performed single-cell RNA sequencing of sorted bone marrow CD34+ hematopoietic stem and progenitor cells (HSPCs) from eight GATA2 deficiency patients, who had various well characterized pathogenic GATA2 mutations and clinically manifest myelodysplasia (Fig A). We characterized transcriptomes in lineages, computationally defined cells with chromosomal abnormalities, and described gene expression of these cells. We first used gene expression signatures of single HSPCs from healthy donors to impute identity of stem cells or progenitors of erythroid/megakaryocytic (MEP), myeloid and lymphoid lineages, and then proceeded to reconstruct hematopoietic differentiation by pseudotemporal ordering. Mapping patients' cells onto normal hematopoiesis, we observed marked deficiency in granulocyte/monocyte/lymphocyte differentiation and relatively preserved MEP (Fig B, top). Based on signature genes of each differentiated lineage, we computed a score of gene expression to corresponding lineage in individual patient and found a significantly lower score for granulocyte/monocyte and B cell progenitors in patients, MEP scores tended to be higher in patients without significance (Fig B, bottom). Upregulated genes in patients' HSPCs related to erythroid differentiation, and cell cycle and proliferation (Fig C), while downregulated gene sets were highly enriched in immune responses (Fig D). Differential co-expressed genes were defined by weighted correlation network analysis; they were enriched in immune response and DNA repair (genes exhibited higher correlation in MTOR and T cell receptor signaling pathway in patients [Fig E]). We observed differential effects of GATA2 mutations in distinct lineages, which could only be resolved with the single cell method in rare cell populations. In HSCs, upregulated genes were related to apoptosis and downregulated genes were involved in maintenance of quiescence and cell cycle; there was increased expression of erythroid/megakaryocytic priming programs and decreased expression of lymphoid priming programs. We also observed lower GATA2 expression levels at initial stages of hematopoiesis, likely contributing to disturbed stem cell homeostasis in GATA2 deficiency. DNA repair genes were downregulated in trisomy 8 cells, possibly rendering these cells vulnerable to second-hit somatic mutations and additional chromosomal abnormalities. Cells with complex cytogenetics had defects in multi-lineage differentiation and cell cycle. In conclusion, germline GATA2 mutations modulate gene expression and change gene coexpression patterns. Distinct lineages show different transcriptional profiles resulting from GATA2 mutations. The prominent deficiency in myeloid/lymphoid lineages in GATA2 deficiency is partly due to expression of aberrant gene programs in HSCs prior to lineage commitment. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Differentiation Profiling of Marrow Stem Cells: A Megakaryopoietic Hotspot and the Continuum Model of Hematopoiesis.

Blood ◽

10.1182/blood.v106.11.4217.4217 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4217-4217

Author(s):

Gerald A. Colvin ◽

Dooner Gerri ◽

Delia Demers ◽

Shiela Pascual ◽

Samuel Chung ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

S Phase ◽

Platelet Glycoprotein ◽

No Production ◽

Colony Stimulating Factor ◽

Hematopoietic Stem ◽

Steel Factor ◽

Stimulating Factor

Abstract Hierarchical models of hematopoiesis suppose an ordered system in which stem cells and progenitors with specific fixed differentiation potentials exist. We show here that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell cycle transit. This along with other data strongly suggest that stem cell regulation is not based on the classic hierarchical model, but instead more on a functional continuum We have previously shown that hematopoietic stem cells reversibly shift their engraftment phenotype with cytokine induced cell cycle transit. Further work has shown that adhesion protein, cytokine receptor, gene expression and progenitor phenotypes also shift. Evolving data indicate the phenotype of murine marrow stem cells reversible change with cell cycle transit. Murine experiments have been performed on highly purified, quiescent G0-1 lineagenegativerhodaminelowHoeschtlow (LRH) marrow stem cells. When exposed to thrombopoietin, FLT3-ligand and steel factor, they synchronously pass through cell cycle as measured by propidium iodide, cell doublings and tritiated thymidine. LRH cells enter S-phase in a synchronized fashion by 18 hours, leave S-phase at 40–42 hours and divide between 44–48 hours. The capacity of these cells to respond to a differentiation inductive signal (granulocyte colony-stimulating factor, granulocyte-macrophage colony stimulating factor and steel factor) is altered at different points in cell cycle. Megakaryocyte production is specifically focused at early to mid S-phase, this returned to baseline before the first cell division. Population based cultures after 14-days of differentiation culture produced up to 49% megakaryocytes with stem cells sub-cultured during early-mid S-phase with little to no production with colonies cultured from stem cells in G0-1 or G2 phase at time of differentiation induction signaling. Cell type was confirmed by staining cells with acetylcholinesterase, antibodies to platelet glycoprotein complex IIb/IIIa and von Willebrand’s factor. Evaluation of gene expression at this hotspot showed a marked increase in expression of CD4 with up to 464.2 fold increase above baseline. Sca-1 and transcriptional factor FOG was strikingly amplified at S-phase as well as other relevant markers. While pertinent cytokine receptors were not increased, studies on a clonal level confirm the existence of a reversible megakaryocytic hotspot. Compared with other time-points relating to cell cycle position prior to differentiation sub-culture in one experiment, 33% of clonally derived colonies that grew from early S-phase cells and 10% of colonies that grew from mid S-phase cells had megakaryocytes present two weeks after initiation of culture compared with 0% for G0-1 and G2 cells. Granulocyte differentiation also showed specific differentiation hotspots, but presentation is outside the scope of this abstract. These data indicate that marrow hematopoiesis stem cells exist in a continuum, not in a hierarchy with continuously changing windows of transcriptional opportunity.

Download Full-text

MicroRNA-29a Is An Oncogene in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v112.11.683.683 ◽

2008 ◽

Vol 112 (11) ◽

pp. 683-683

Author(s):

Christopher Y. Park ◽

Yoon-Chi Han ◽

Govind Bhagat ◽

Jian-Bing Fan ◽

Irving L Weissman ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Acute Myeloid Leukemia ◽

Hematopoietic Stem Cells ◽

Mirna Expression ◽

Myeloid Leukemia ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract microRNAs (miRNAs) are short, non-protein encoding RNAs that bind to the 3′UTR’s of target mRNAs and negatively regulate gene expression by facilitating mRNA degradation or translational inhibition. Aberrant miRNA expression is well-documented in both solid and hematopoietic malignancies, and a number of recent miRNA profiling studies have identified miRNAs associated with specific human acute myeloid leukemia (AML) cytogenetic groups as well as miRNAs that may prognosticate clinical outcomes in AML patients. Unfortunately, these studies do not directly address the functional role of miRNAs in AML. In fact, there is no direct functional evidence that miRNAs are required for AML development or maintenance. Herein, we report on our recent efforts to elucidate the role of miRNAs in AML stem cells. miRNA expression profiling of AML stem cells and their normal counterparts, hematopoietic stem cells (HSC) and committed progenitors, reveals that miR-29a is highly expressed in human hematopoietic stem cells (HSC) and human AML relative to normal committed progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors is sufficient to induce a myeloproliferative disorder (MPD) that progresses to AML. During the MPD phase of the disease, miR-29a alters the composition of committed myeloid progenitors, significantly expedites cell cycle progression, and promotes proliferation of hematopoietic progenitors at the level of the multipotent progenitor (MPP). These changes are manifested pathologically by marked granulocytic and megakaryocytic hyperplasia with hepatosplenomegaly. Mice with miR-29a-induced MPD uniformly progress to an AML that contains a leukemia stem cell (LSC) population that can serially transplant disease with as few as 20 purified LSC. Gene expression analysis reveals multiple tumor suppressors and cell cycle regulators downregulated in miR-29a expressing cells compared to wild type. We have demonstrated that one of these genes, Hbp1, is a bona fide miR-29a target, but knockdown of Hbp1 in vivo does not recapitulate the miR-29a phenotype. These data indicate that additional genes are required for miR-29a’s leukemogenic activity. In summary, our data demonstrate that miR-29a regulates early events in normal hematopoiesis and promotes myeloid differentiation and expansion. Moreover, they establish that misexpression of a single miRNA is sufficient to drive leukemogenesis, suggesting that therapeutic targeting of miRNAs may be an effective means of treating myeloid leukemias.

Download Full-text

Hematopoiesis in the Elderly: Age-Associated Effects in Frequency, Function, and Gene Expression of Human Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.1505.1505 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1505-1505

Author(s):

Wendy W. Pang ◽

Elizabeth A. Price ◽

Irving L. Weissman ◽

Stanley L. Schrier

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Expression Profiles ◽

Frequency Function ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cell Cycle Status

Abstract Abstract 1505 Poster Board I-528 Aging of the human hematopoietic system is associated with an increase in the development of anemia, myeloid malignancies, and decreased adaptive immune function. While the hematopoietic stem cell (HSC) population in mouse has been shown to change both quantitatively as well as functionally with age, age-associated alterations in the human HSC and progenitor cell populations have not been characterized. In order to elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated and compared HSC and other hematopoietic progenitor populations prospectively isolated via fluorescence activated cell sorting (FACS) from 10 healthy young (20-35 years of age) and 8 healthy elderly (65+ years of age) human bone marrow samples. Bone marrow was obtained from hematologically normal young and old volunteers, under a protocol approved by the Stanford Institutional Review Board. We determined by flow cytometry the distribution frequencies and cell cycle status of HSC and progenitor populations. We also analyzed the in vitro function and generated gene expression profiles of the sorted HSC and progenitor populations. We found that bone marrow samples obtained from normal elderly adults contain ∼2-3 times the frequency of immunophenotypic HSC (Lin-CD34+CD38-CD90+) compared to bone marrow obtained from normal young adults (p < 0.02). Furthermore, upon evaluation of cell cycle status using RNA (Pyronin-Y) and DNA (Hoechst 33342) dyes, we observed that a greater percentage of HSC from young bone marrow are in the quiescent G0- phase of the cell cycle compared to elderly HSC, of which there is a greater percentage in G1-, S-, G2-, or M-phases of the cell cycle (2.5-fold difference; p < 0.03). In contrast to the increase in HSC frequency, we did not detect any significant differences in the frequency of the earliest immunophenotypic common myeloid progenitors (CMP; Lin-CD34+CD38+CD123+CD45RA-), granulocyte-macrophage progenitors (GMP; Lin-CD34+CD38+CD123+CD45RA+), and megakaryocytic-erythroid progenitors (MEP; Lin-CD34+CD38+CD123-CD45RA-) from young and elderly bone marrow. We next analyzed the ability of young and elderly HSC to differentiate into myeloid and lymphoid lineages in vitro. We found that elderly HSC exhibit diminished capacity to differentiate into lymphoid B-lineage cells in the AC6.21 culture environment. We did not, however, observe significant differences in the ability of young and elderly HSC to form myeloid and erythroid colonies in methylcellulose culture, indicating that myelo-erythroid differentiation capacity is preserved in elderly HSC. Correspondingly, gene expression profiling of young and elderly human HSC indicate that elderly HSC have up-regulation of genes that specify myelo-erythroid fate and function and down-regulation of genes associated with lymphopoiesis. Additionally, elderly HSC exhibit increased levels of transcripts associated with transcription, active cell-cycle, cell growth and proliferation, and cell death. These data suggest that hematopoietic aging is associated with intrinsic changes in the gene expression of human HSC that reflect the quantitative and functional alterations of HSC seen in elderly bone marrow. In aged individuals, HSC are more numerous and, as a population, are more myeloid biased than young HSC, which are more balanced in lymphoid and myeloid potential. We are currently investigating the causes of and mechanisms behind these highly specific age-associated changes in human HSC. Disclosures: Weissman: Amgen: Equity Ownership; Cellerant Inc.: ; Stem Cells Inc.: ; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

Download Full-text

Loss Of p53 Rescues The Defective Function Of Foxo3-/- Hematopoietic Stem Cells But Enhances Their Predisposition To Malignancy

Blood ◽

10.1182/blood.v122.21.4199.4199 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4199-4199 ◽

Cited By ~ 1

Author(s):

Carolina L. Bigarella ◽

Pauline Rimmele ◽

Rebeca Dieguez-Gonzalez ◽

Raymond Liang ◽

Brigitte Izac ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Dna Repair ◽

Stem Cell ◽

Gene Expression Profile ◽

Double Mutant ◽

Self Renewal ◽

Hematopoietic Stem ◽

Anti Oxidant

Abstract Leukemic stem cells (LSCs) share many of the same properties of normal hematopoietic stem cells (HSCs) including their highly quiescent state, capacity to self-renew, low levels of reactive oxygen species (ROS) and enhanced DNA repair program. These properties make the efficient and specific eradication of these cells challenging. Foxo3 and p53 are two transcription factors essential for the modulation of HSC quiescence and self-renewal. While Foxo3 is inhibited by signaling from several oncoproteins but crucial for the maintenance of the LSCs in both chronic and acute myeloid leukemia (CML and AML respectively), mutations of p53 although rare, are associated with poor prognosis in advanced stages of these diseases. In vivo ROS-mediated activation of p53 is known to lead to loss of quiescence, alterations of cell cycle and exhaustion of the Foxo3-/- HSC pool. Seeking to understand the contribution of p53 to Foxo3-/- HSC cycling defects, we crossed p53+/- and Foxo3+/- mice. To our surprise we found the bone marrow (BM) frequency of both p53+/-Foxo3-/- and p53-/-Foxo3-/- LSK (Lin-Sca1+cKit+) and long-term-HSC (LT-HSC, LSK Flk2-CD34-) populations greatly increased as compared to their Foxo3-/- counterparts (n=5 mice per genotype; p<0.05). Using Ki67 and DAPI staining we found that loss of one or both alleles of p53 gradually rescued the cell cycle defect of Foxo3-/- HSC and increased the frequency of LSK cells in Go by 2-fold. Loss of p53 also rescued the impaired capacity of Foxo3-/- LSK cells to competitively repopulate multilineage blood over 16 weeks, as shown by the higher frequency of p53+/-Foxo3-/- and p53-/-Foxo3-/- donor-derived cells in the peripheral blood of recipient animals (∼47% recipients of double-mutant cells versus 20% in Foxo3-/- recipients, n=5 per group). Furthermore, loss of p53 significantly improved the compromised self-renewal of Foxo3 mutant HSC in serial BM transplantations. In our quest to identify mechanisms whereby p53 depletion improves Foxo3-/- HSC function, we noticed that the DNA damage accumulated in Foxo3-/- HSC at the steady-state was remarkably ameliorated by removal of one or both alleles of p53 from Foxo3-/- HSCs, as measured by flow cytometry levels of phospho-H2AX (gamma-H2AX) and DNA breaks by comet assay (n=3, p<0.05). Unexpectedly, ROS levels were also significantly reduced by 30% in p53+/-Foxo3-/- in comparison to Foxo3-/- LSK cells, while ROS levels in p53+/- LSK cells were similar to that in WT cells. Consistent with these results, the expression of several anti-oxidant enzymes including Sod1, Sod2, Catalase, Gpx1, Sesn1 and Sesn2 (n≥2), was highly upregulated while a number of genes implicated in mitochondrial generation of ROS were significantly deregulated as a result of loss of one or both alleles of p53. These combined findings suggest that a switch from anti-oxidant to pro-oxidant activity of p53 contributes to Foxo3-/- HSC defects. Despite their apparent normal stem cell function, p53+/-Foxo3-/- HSC were highly altered in their gene expression profile. Interestingly, Gene Set Enrichment Analysis (GSEA) of the microarray analysis (Illumina bead chip mouse-Ref8) of WT, p53+/-, Foxo3-/-, and p53+/-Foxo3-/- LSK cells showed that a cluster of genes associated with fatty acid metabolism was highly enriched in p53+/-Foxo3-/- HSCs (ES=0.746; p<0.01). In addition, from 3976 genes exclusively deregulated in p53+/-Foxo3-/- LSK cells, 201 (out of 1051) overlapped with genes downregulated, while 9 (out of 14) overlapped with genes exclusively upregulated in a LSC-gene signature. To evaluate whether this pre-leukemic profile was associated with increased susceptibility to malignancy, we compared the potential and timeline of BCR-ABL-transformed p53+/-Foxo3-/- HSC as compared to controls in establishing CML in mice. We found a shorter time to the onset of the disease and decreased survival of the recipients of p53+/-Foxo3-/- transformed HSCs (n=4 per group, p<0.05) as compared to WT and Foxo3-/- controls. We propose that the p53+/-Foxo3-/- double-mutant HSCs are enriched for preleukemic stem cells based on their quiescence and self-renewal capacity, low ROS, robust DNA repair, susceptibility to transformation and aberrant gene expression profile. These findings raise the possibility that the coordinated Foxo3 and p53 regulation of ROS wires together the stem cell program. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Radiation-induced, cell cycle-related gene expression in aging hematopoietic stem cells: enigma of their recovery

Annals of the New York Academy of Sciences ◽

10.1111/nyas.12401 ◽

2014 ◽

Vol 1310 (1) ◽

pp. 69-73 ◽

Cited By ~ 6

Author(s):

Yoko Hirabayashi

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Related Gene ◽

Hematopoietic Stem ◽

Cell Cycle Related Gene ◽

Radiation Induced

Download Full-text

Single Cell Transcriptome Analysis of GATA2 Deficiency in Hematopoiesis Modeled with Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood-2018-99-118410 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5087-5087

Author(s):

Francis Guitart ◽

Moonjung Jung ◽

Stefan Cordes ◽

Shiqin Yu ◽

Jizhong Zou ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Single Cell ◽

Differential Gene Expression ◽

Hematopoietic Stem ◽

Manifold Alignment ◽

Gata2 Deficiency ◽

Differential Gene ◽

Cell Transcriptome ◽

Single Cell Transcriptome

Abstract GATA2 deficiency is a rare, inherited or sporadic genetic disorder characterized by variable onset of a pleomorphic constellation of immune, hematologic and lymphatic abnormalities linked to heterozygous mutations in the Gata2 gene. Patients develop monocyte, B cell, NK cell and dendritic cell deficiencies resulting in vulnerabilities to unusual infections. Patients with GATA2 deficiency also frequently progress to bone marrow failure, myelodysplastic syndrome and/or acute myelogenous leukemia. GATA proteins are transcription factors with central roles in early embryonic development and lineage specification. GATA2 is a master regulator of hematopoiesis, implicated in the initial generation and maintenance of hematopoietic stem cells (HSC). Murine models recapitulate the human phenotype incompletely: GATA2 heterozygous knockout mice do not manifest loss of monocyte, B cells or NK cells; however, serial repopulation assays show decreased engraftment potential. Direct studies of primary HSC from patients with GATA2 deficiency are challenging due to the generally hypocellular marrow. We hypothesized that human pluripotent stem cells, particularly patient-specific iPSC, could be used to study potential developmental defects in GATA2 deficiency, overcoming a lack of primary HSC. In order to gain insights into the impact of human GATA2 deficiency on hematopoietic differentiation, we compared the single cell transcriptomes of HSPC differentiated from (i) iPSCs from a patient with GATA2 deficiency due to a mutation p.R337X (c.1009C>T) (ii) isogenic iPSCs created via homology-directed repair of Gata2 p.R337X, (iii) iPSCs from a healthy control and (iv) isogenic Gata2 heterozygous mutant iPSCs with monoallelic frameshift mutations in the second zinc finger domain. Mesodermal and hematopoietic differentiation was performed under feeder-free, defined media conditions. At day 0, iPSCs were plated in mesodermal induction media containing VEGF, SCF, Activin A and Y27632 in STEMdiff APEL media. Mesodermal induction was continued until day 4, when embryoid bodies were cultured in hematopoietic specification media with SCF, FLT3L, IL3, IL6, G-CSF and BMP4 until day 16, when CD34+CD45+ iPSC-derived hematopoietic stem and progenitor cells (iHSC) were enumerated and sorted by fluorescence-activated cell sorting. Single cell RNA-seq was performed using the 10XGenomics Chromium platform and primary analysis via CellRanger. Scater was used to filter outlier cells. Seurat as used to compute multiple manifold alignment and differential gene expression. Cell classification, pseudotemporal ordering and branch point analysis were performed with monocle. URD was used to calculate confirmatory diffusion maps and pseudotemporal ordering. We analyzed 7,855 iHSPC (2952 from GATA2-deficient patient, 241 isogenic iHSPCs after repair of Gata2 mutation, 2,605 from a healthy volunteer and 2,057 from isogenic heterozygous Gata2 knockout iHSPCs) after filtering of outliers. We computed multiple manifold alignment to mitigate batch effects. Differential gene expression across Gata2 mutation status found that 42 out of 102 (42%) target genes of GATA2 (c.f. TRANSFAC database of curated transcription factor targets) were differentially expressed with adjusted p-values less than 0.05. Semi-supervised classification of cell-types and pseudotemporal ordering via monocle revealed two branch points, consistent with developmental branchings at the level of CLP and CMP multipotent progenitors. The numbers of cells along each branch was found to be statistically different (χ2=30.07, p-value = 3e-7) with the biggest differences noted in the lymphoid branch (state 4). Differential gene expression in this branch revealed a differential up-regulation of Notch1, CD69 and FKBPs and differential down-regulation of CD14. In conclusion, iPSC/iHSPC differentiation models combined with single cell transcriptome analysis may be a valuable tool to identify pathways responsible for impaired hematopoietic/lymphatic development in GATA2 deficiency. Figure. Figure. Disclosures Dunbar: National Institute of Health: Research Funding. Winkler:National Institute of Health: Research Funding.

Download Full-text

Hoxa9+Meis1a Efficiently Transform Hematopoietic Stem Cells but Not Committed Progenitors.

Blood ◽

10.1182/blood.v110.11.3375.3375 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3375-3375

Author(s):

Yingzi Wang ◽

Andrei Krivtsov ◽

Grigoriy Losyev ◽

Scott A. Armstrong

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

High Efficiency ◽

Single Cells ◽

Global Gene Expression ◽

Self Renewal ◽

Hematopoietic Stem ◽

Clonogenic Potential

Abstract The homeobox transcription factors Hoxa9 and Meis1a induce the expansion of hematopoietic stem cells (HSC) and cooperate to induce acute myeloid leukemia (AML). We have recently shown that Hoxa9 and Meis1a are part of a self-renewal program found in leukemia stem cells (LSC) isolated from murine leukemias initiated by expression of MLL-AF9 in committed granulocyte macrophage progenitors (GMP). However, it remains unclear whether the Hoxa9-Meis1a complex is sufficient to mimic the leukemogenic effects of MLL-AF9. Therefore we assessed the clonogenic and leukemogenic activity of HSC and GMP transduced with Hoxa9-Meis1a and compared this to MLL-AF9 transduced HSC and GMP. We expressed either Hoxa9-Meis1a or MLL-AF9 in HSC or GMP and sorted single cells into 96 well plates. The data demonstrate that the clonogenic potential of single HSC or GMP expressing Hoxa9-Meis1a or MLL-AF9 are similar and both produce cells that can be replated in vitro for greater than 6 weeks. Remarkably, Hoxa9-Meis1a induces leukemia with high efficiency when expressed in HSC but not committed GMP, while MLL-AF9 can fully transform both HSC and GMP. Next, we identified a population of cells from Hoxa9-Meis1a leukemias that were enriched for LSC. Even though the leukemias were initiated from HSC, the LSC population possessed an immunophenotype and global gene expression program more consistent with differentiated myeloid cells. Further characterization of gene expression in the Hoxa9-Meis1a LSC found that only a subset of the MLL-AF9 self-renewal signature is activated by Hoxa9-Meis1a. These findings show that Hoxa9-Meis1a can efficiently induce LSC from HSC but not GMP. This suggests Hoxa9 and Meis1a induced leukemogenesis is dependent upon cellular context and require programs/pathways active in HSC in order to initiate leukemia. Thus MLL-AF9 must activate pathways critical for LSC development in addition to Hoxa9-Meis1a in order to fully transform committed progenitor cells. Identification of these cooperating pathways should provide insight into MLL-rearranged and other AML.

Download Full-text