Comprehensive Analysis Of HOX Gene Expression and DNA Methylation From 189 Primary AMLs Demonstrates Canonical Patterns Associated With Hematopoietic Stem/Progenitors and Recurrent AML Mutations

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2496-2496 ◽  
Author(s):  
David H Spencer ◽  
Margaret A. Young ◽  
Jeffery M. Klco ◽  
Timothy J. Ley
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Hox Genes ◽  
Embryonic Stem ◽  
Normal Karyotype ◽  
Hematopoietic Stem ◽  
Hox Gene ◽  
Cd34 Cells ◽  
Methylation Patterns

Abstract HOX genes encode a family of homeodomain transcription factors with important roles in hematopoiesis. Expression of HOX genes is also a common feature of acute myeloid leukemia (AML), and functional studies have suggested that HOX-dependent pathways may contribute to leukemogenesis. Although HOX expression is known to correlate with specific AML mutations, the patterns of expression of all 39 HOX genes in primary AML samples, and their relationships with recurrent AML mutations, are incompletely understood. In addition, little is known about the influence of AML mutations on DNA methylation at the HOX loci, and the relationship between HOX gene expression and methylation in AML. In this study, we carried out a combined analysis of gene expression data from microarray and RNA-sequencing platforms and genome-wide DNA array-based methylation from 189 primary AML samples that have been previously characterized by either whole-genome or whole exome sequencing. We also measured expression and methylation using the same platforms from normal bone marrow subsets, including CD34+ cells, promyelocytes, monocytes, neutrophils and lymphocytes, and obtained expression data from CD34+ hematopoietic precursors generated from in vitro differentiation of human embryonic stem cells. Our analysis confirmed previous work on the general patterns of HOX expression in AML. The HOXA and HOXB genes showed variation both within each cluster and across the AMLs, although high level expression was restricted to a subset of these genes, including HOXA3, HOXA5, HOXA7, HOXA9, HOXA10, HOXB2-HOXB4, and HOXB6, as well as HOX cofactor MEIS1; HOXC and HOXD genes were minimally expressed in all of the samples. These observations were orthogonally validated by RNA-seq, and with a targeted Nanostring expression platform. Consistent with previous studies, MLL-positive AML samples (n=11) expressed only HOXA genes and MEIS1. AML samples with CBFB-MYH11 rearrangements (n=12) showed expression of only MEIS1, and HOXB2-HOXB4 at moderate levels; RUNX1-RUNX1T1 (n=7) and PML-RARA (n=19) samples did not detectably express any HOX genes. In AMLs with a normal karyotype (n=85), we observed two distinct patterns; one pattern displayed little or no HOX gene expression (7/85; 8%), and another displayed canonical expression of a specific subset of the HOXA and HOXB genes and MEIS1 (78/85; 92%) with similar relative HOX gene expression levels in all cases. Comparison of this pattern with normal bone marrow revealed the same HOX expression pattern in normal CD34+ cells; additional analysis showed that this pattern was confined to hematopoietic stem/progenitor cells, but was not seen in more mature cells, including other CD34+ subsets, promyelocytes, monocytes and neutrophils. We also measured HOX gene expression in CD34+ hematopoietic precursors generated from in vitro differentiation of human embryonic stem cells, which revealed expression of only MEIS1 and the canonical HOXB genes, suggesting that activation of these genes may represent the earliest events in the HOX pathway of hematopoietic development. Correlation of HOX expression with recurrent AML mutations by gene set enrichment analysis demonstrated a significant association with NPM1 (P<10-4) and DNMT3A (P<10-2) mutations, but not with other recurrent somatic mutations, including FLT3,IDH1/IDH2, and TET2. Methylation at the HOX loci demonstrated patterns that correlated with HOX expression, including hypomethylation at HOX promoters in samples with high expression. However, additional mutation-specific patterns were apparent. For example, NPM1-mutant AMLs demonstrated a distinct methylation pattern that included hypomethylation at the HOXB3 promoter, which was not shared with CBFB-MYH11 cases or other AMLs with HOXB3 expression. In summary, our comprehensive analysis demonstrates canonical expression and methylation patterns at the HOX loci in AML. These patterns correspond to specific recurrent AML mutations, and the dominant pattern in most normal karyotype AMLs mimics the signature of hematopoietic stem cells. This supports previous observations of developmental regulation of HOX genes in hematopoiesis, and implies that this normal stem cell signature is “captured” in the majority of AMLs with normal karyotype. In addition, distinct methylation patterns at HOX loci suggest that multiple regulatory mechanisms are involved in HOX expression in AML. Disclosures: No relevant conflicts of interest to declare.

LYL1, Class II Basic Helix-Loop-Helix Transcription Factor, Induces the Differentiation of Common Marmoset Embryonic Stem Cells to Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2348-2348
Author(s):  
Hirotaka Kawano ◽  
Tomotoshi Marumoto ◽  
Michiyo Okada ◽  
Tomoko Inoue ◽  
Takenobu Nii ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Lymphoblastic Leukemia ◽  
Embryonic Stem ◽  
Common Marmoset ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Cd34 Cells

Abstract Abstract 2348 Since the successful establishment of human embryonic stem cells (ESCs) in 1998, transplantation of functional cells differentiated from ESCs to the specific impaired organ has been expected to cure its defective function [Thomson JA et al., Science 282:1145–47, 1998]. For the establishment of the regenerative medicine using ESCs, the preclinical studies utilizing animal model systems including non-human primates are essential. We have demonstrated that non-human primate of common marmoset (CM) is a suitable experimental animal for the preclinical studies of hematopoietic stem cells (HSCs) therapy [Hibino H et al., Blood 93:2839–48, 1999]. Since then we have continuously investigated the in vitro and in vivo differentiation of CM ESCs to hematopoietic cells by the exogenous hematopoietic gene transfer. In earlier study, we showed that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs is promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., Stem Cells 24:2014-22,2006]. However those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene is not enough to induce functional HSCs which have self-renewal capability and multipotency. Thus we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation from ESCs to HSCs, based on the comparison of gene expression level between human ESCs and HSCs by Digital Differential Display from the Uni-Gene database at the NCBI web site (http://www.ncbi.nlm.nih.gov/UniGene/). Then, we transduced the respective candidate gene in CM ESCs (Cj11), and performed embryoid body (EB) formation assay to induce their differentiation to HSCs for 9 days. We found that lentiviral transduction of LYL1, a basic helix-loop-helix transcription factor, in EBs derived from Cj11, one of CM ESC lines, markedly increased the number of cells positive for CD34, a marker for hematopoietic stem/progenitors. The lymphoblastic leukemia 1 (LYL1) was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., Cell 58:77-83.1989]. These class II bHLH transcription factors regulate gene expression by binding to target gene sequences as heterodimers with E-proteins, in association with Gata1 and Gata2 [Goldfarb AN et al., Blood 85:465-71.1995][Hofmann T et al., Oncogene 13:617-24.1996][Hsu HL et al., Proc Natl Acad Sci USA 91:5947-51.1994]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., Blood 107:4678-4686. 2006]. And, overexpression of Lyl1 in mouse bone marrow cells induced the increase of HSCs, HPCs and lymphocytes in vitro and in vivo [Lukov GL et al., Leuk Res 35:405-12. 2011]. These information indicate that LYL1 plays important roles in hematopoietic differentiation in primate animals including human and common marmoset. To examine whether overexpression of LYL1 in EBs can promote hematopoietic differentiation in vitro we performed colony-forming unit (CFU) assay, and found that LYL1-overexpressing EBs showed the formation of multi-lineage blood cells consisting of erythroid cells, granulocytes and macrophages. Next, we analyzed gene expression level by RT-PCR, and found that the transduction of LYL1 induced the expression of various hematopoietic genes. These results suggested that the overexpression of LYL1 can promote the differentiation of CM ESCs to HSCs in vitro. Furthermore we found that the combined overexpression of TAL1 and LYL1 could enhance the differentiation of CD34+ cells from CM ESCs than the respective overexrpession of TAL1 or LYL1. Collectively, our novel technology to differentiate hematopoietic cells from ESCs by the transduction of specific transcription factors is novel, and might be applicable to expand human hematopoietic stem/progenitor cells in vitro for future regenerative medicine to cure human hematopoietic cell dyscrasias. Disclosures: No relevant conflicts of interest to declare.

BMP4 Favors Hematopoietic Differentiation from Murine Embryonic Stem Cells and Acts by Activation of the cdx-hox Pathway.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3613-3613
Author(s):  
Claudia Lengerke ◽  
Yuan Wang ◽  
Frank Yates ◽  
Leila Maouche-Chretien ◽  
George Q. Daley
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hox Genes ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Hematopoietic Stem ◽  
Hox Gene ◽  
Murine Embryonic Stem Cells

Abstract Cdx4 and cdx1, members of the caudal family of homeodomain-containing transcriptional regulators, are important for specifying the hematopoietic fate of mesoderm in the zebrafish. We have shown that the cdx4 gene plays a role in enhancing hematopoietic fate during in vitro differentiation of murine ESCs (Davidson et al., Nature 2003). Cdx4 induces hox genes, and genetic modification of mESCs with a combination of cdx4 and hoxb4 promotes long-term engraftment of ESC-derived HSCs in lethally irradiated primary and secondary mice (Wang et al, submitted). While cdx1 is known to be a direct target of signaling by the embryonic morphogens fgf, wnt3a, and retinoids, morphogens acting upstream of cdx4 have not yet been defined. Our goal is to determine optimal morphogen conditions for hematopoietic commitment from murine embryonic stem cells by evaluating activation of the cdx-hox pathway. We have developed quantitative RT-PCR assays for the cdx genes (cdx4, cdx1 and cdx2) and multiple hox genes as well as markers specific to hematopoietic stem cells and lineages. We have used these assays, together with a reporter line engineered to express GFP from the brachury locus (Fehling et al., Development 2003), to characterize the conditions for mesodermal induction and hematopoietic fate specification following addition of morphogens to differentiating cultures of ES cells under serum-free conditions. Among all morphogens tested (BMP4, activin, nodal, wnt3a, wnt5a, sonic hedgehog, indian hedgehog, retinoic acid), only BMP4 has been found to strongly induce CDX4 gene expression within the developing embryoid bodies, while addition of the BMP4 inhibitor noggin to serum suppressed CDX4 expression. Addition of BMP4 significantly increases the number of emerging CD41+ and CD45+ cells, the precursors of definitive hematopoietic stem cells. We are currently analyzing the functional changes following BMP4 exposure, and correlating hematopoietic maturation with changes in the Hox gene expression pattern. Analysis of the cdx-hox gene pathway provides a means of otpimizing induction of hematopoietic fate by application of embryonic morphogens.

Induced Hematopoietic Differentiation Of Common Marmoset Embryonic Stem Cells By LYL1 Can Give Rise To Hematopoietic Stem Cells Having The Enhanced Bone Marrow Reconstitution Capability

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1192-1192
Author(s):  
Hirotaka Kawano ◽  
Tomotoshi Marumoto ◽  
Takafumi Hiramoto ◽  
Michiyo Okada ◽  
Tomoko Inoue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Lymphoblastic Leukemia ◽  
Embryonic Stem ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Hsc Transplantation

Abstract Hematopoietic stem cell (HSC) transplantation is the most successful cellular therapy for the malignant hematopoietic diseases such as leukemia, and early recovery of host’s hematopoiesis after HSC transplantation has eagerly been expected to reduce the regimen related toxicity for many years. For the establishment of the safer and more efficient cell source for allogeneic or autologous HSC transplantation, HSCs differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that show indefinite proliferation in an undifferentiated state and pluripotency, are considered to be one of the best candidates. Unfortunately, despite many recent efforts, the HSC-specific differentiation from ESCs and iPSCs remains poor [Kaufman, DS et al., 2001][Ledran MH et al., 2008]. In this study, we developed the new method to differentiate HSC from non-human primate ESC/iPSC. It has been reported that common marmoset (CM), a non-human primate, is a suitable experimental animal for the preclinical studies of HSC therapy [Hibino H et al., 1999]. We have been investigated the hematopoietic differentiation of CM ESCs into HSCs, and previously reported that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs were promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., 2006]. However, those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene was not sufficient to induce functional HSCs which have self-renewal capability and multipotency. Thus, we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation of ESCs into HSCs, based on the previous study of hematopoietic differentiation from human and mouse ESCs. And CM ESCs (Cj11) lentivirally transduced with the respective candidate gene were processed for embryoid body (EB) formation to induce their differentiation into HSCs for 9 days. We found that lentiviral transduction of LYL1 (lymphoblastic leukemia 1), a basic helix-loop-helix transcription factor, in EBs markedly increased the proportion of cells positive for CD34 (approximately 20% of LYL1-transduced cells). RT-PCR showed that LYL1-transduced EBs expressed various hematopoietic genes, such as TAL1, RUNX1 and c-KIT. To examine whether these CD34+ cells have the ability to differentiate into hematopoietic cells in vitro, we performed colony-forming unit (CFU) assay, and found that CD34+ cells in LYL1-transduced EBs could form multi-lineage blood colonies. Furthermore the number of blood colonies originated from CD34+CD45+ cells in LYL1-transduced EBs was almost the same as that from CD34+CD45+ cells derived from CM bone marrow. These results suggested that enforced expression of LYL1 in CM ESCs promoted the emergence of HSCs by EB formation in vitro. The LYL1 was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., 1989]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., 2006]. And, transduction of Lyl1 in mouse bone marrow cells induced the increase of HSCs and lymphocytes in vitro and in vivo [Lukov GL et al., 2011]. Therefore we hypothesized that LYL1 may play essential roles in bone marrow reconstitution by HSCs differentiated from CM ESCs. To examine this, we transplanted CD34+ cells derived from LYL1-transduced CM ESCs into bone marrow of sublethally irradiated NOG mice, and found that about 7% of CD45+ cells derived from CM ESCs were detected in peripheral blood (PB) of recipient mice at 8 weeks after transplant (n=4). Although CM CD45+ cells disappeared at 12 weeks after transplant, CD34+ cells (about 3%) were still found in bone marrow at the same time point. Given that TAL1-transduced EBs derived from CM ESCs could not reconstitute bone marrow of irradiated mice at all, LYL1 rather than TAL1 might be a more appropriate transcription factor that can give rise to CD34+ HSCs having the enhanced capability of bone marrow reconstitution from CM ESCs. We are planning to do in vivo study to prove this hypothesis in CM. Disclosures: No relevant conflicts of interest to declare.

Derivation of Hematopoietic Stem Cells from Embryonic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 223-223 ◽  
Author(s):  
Yuan Wang ◽  
Frank Yates ◽  
Eugenia Dikovskaia ◽  
Patricia Ernst ◽  
Alan J. Davidson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hox Genes ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Lymphoid Cells ◽  
Time Interval ◽  
Hematopoietic Stem

Abstract Despite the significant in vitro blood-forming potential of murine embryonic stem cells (ESCs), deriving hematopoietic stem cells (HSCs) that can reconstitute irradiated mice has proven to be challenging. Previously, we successfully engrafted lethally irradiated adult mice with ESCs engineered to ectopically express the homeodomain gene hoxB4. In engrafted animals, blood reconstitution showed a myeloid predominance, likely due to an inability to fully pattern the adult HSC from these embryonic populations. Recently, we have investigated cdx4, a caudal-related homeobox gene whose function has been linked to blood development in the zebrafish. During in vitro differentiation of murine ESCs, cdx4 is expressed during a very narrow time interval on day 3, coincident with the specification of hematopoietic mesoderm. To further characterize the function of cdx4 in mouse hematopoiesis, we have established a tetracycline-inducible murine embryonic stem cell line. When cdx4 expression is conditionally induced over a protracted period from day 2 and 6, we observe a marked enhancement of hemangioblast formation as well as significant increases in primitive and definitive hematopoietic colonies. Cdx4 acts to induce a broad array of hox genes, including a modest elevation in hoxb4. Co-expression of cdx4 and hoxb4 promotes robust expansion of hematopoietic blasts on supportive OP9 stromal cultures. When injected intravenously into lethally-irradiated mice, these cell populations provide robust radio-protection, and reconstitute high-level lymphoid-myeloid donor chimerism. Marrow from engrafted primary animals can be transplanted into irradiated secondary mice. B220+ splenic lymphoid cells and Mac-1/Gr-1+ marrow myeloid cells purified from primary and secondary mice show multiple common sites of retroviral integration, thereby proving the derivation of long-term hematopoietic stem cells from embryonic stem cells in vitro. Our data support a central role for the cdx4-hox gene pathway in specifying murine HSC development, and establish a robust system for hematopoietic reconstitution from ESCs. We have coupled techniques for generating ESCs by nuclear transfer with these methods for blood reconstitution to model the treatment of genetic disorders of the bone marrow.

scholarly journals Designer blood: creating hematopoietic lineages from embryonic stem cells

Blood ◽  
2006 ◽  
Vol 107 (4) ◽  
pp. 1265-1275 ◽  
Author(s):  
Abby L. Olsen ◽  
David L. Stachura ◽  
Mitchell J. Weiss
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Basic Research ◽  
Cell Types ◽  
Patient Specific ◽  
Es Cell ◽  
Blood Disorders ◽  
Hematopoietic Stem

Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any differentiated tissue upon appropriate manipulation in culture, a property that has been beneficial for studies of hematopoiesis. Until recently, the majority of this work used murine ES cells for basic research to elucidate fundamental properties of blood-cell development and establish methods to derive specific mature lineages. Now, the advent of human ES cells sets the stage for more applied pursuits to generate transplantable cells for treating blood disorders. Current efforts are directed toward adapting in vitro hematopoietic differentiation methods developed for murine ES cells to human lines, identifying the key interspecies differences in biologic properties of ES cells, and generating ES cell-derived hematopoietic stem cells that are competent to repopulate adult hosts. The ultimate medical goal is to create patient-specific and generic ES cell lines that can be expanded in vitro, genetically altered, and differentiated into cell types that can be used to treat hematopoietic diseases.

scholarly journals B-1 lymphoid cells develop independently of Notch signaling during mouse embryonic development

2020 ◽  
Author(s):  
Nathalia Azevedo ◽  
Elisa Bertesago ◽  
Ismail Ismailoglu ◽  
Michael Kyba ◽  
Michihiro Kobayashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Development ◽  
Blood Cell ◽  
Notch Signaling ◽  
Embryonic Stem ◽  
Cell Types ◽  
Lymphoid Cells ◽  
Lineage Specification ◽  
Hematopoietic Stem

AbstractThe in vitro generation from pluripotent stem cells (PSCs) of different blood cell types, in particular those that are not replenished by hematopoietic stem cells (HSCs) like fetal-derived tissue-resident macrophages and innate-like lymphocytes, is of a particular interest. In order to succeed in this endeavor, a thorough understanding of the pathway interplay promoting lineage specification for the different blood cell types is needed. Notch signaling is essential for the HSC generation and their derivatives, but its requirement for tissue-resident immune cells is unknown. Using mouse embryonic stem cells (mESCs) to recapitulate murine embryonic development, we have studied the requirement for Notch signaling during the earliest B-lymphopoiesis and found that Rbpj-deficient mESCs are able to generate B-1 cells. Their Notch-independence was confirmed in ex vivo experiments using Rbpj-deficient embryos. In addition, we found that upregulation of Notch signaling was needed for the emergence of B-2 lymphoid cells. Taken together, these findings indicate that control of Notch signaling dosage is critical for the different B-cell lineage specification and provides pivotal information for their in vitro generation from PSCs for therapeutic applications.

Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2740-2749 ◽  
Author(s):  
CD Helgason ◽  
G Sauvageau ◽  
HJ Lawrence ◽  
C Largman ◽  
RK Humphries
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Homeobox Genes ◽  
Embryoid Bodies ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Differentiation And Proliferation

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events.

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1759-1768 ◽  
Author(s):  
Bernhard Schiedlmeier ◽  
Hannes Klump ◽  
Elke Will ◽  
Gökhan Arman-Kalcek ◽  
Zhixiong Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Therapeutic Window ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Cd34 Cells ◽  
Cord Blood Cd34 ◽  
The Impact

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice or in competition with control vector–transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P = .01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P < .03) and in vivo (P = .01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P < .01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials.

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