Development of Lymphohematopoietic Progenitors during Human Embryonic Stem (hES) Cell Differentiation on OP9 Stromal Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2789-2789
Author(s):  
Igor I. Slukvin ◽  
Maxim A. Vodyanik ◽  
Jack A. Bork ◽  
James A. Thomson
Keyword(s):  
Endothelial Cells ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
Stromal Cells ◽  
Embryonic Stem ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Development ◽  
Cd34 Cells ◽  
Transient Population ◽  
Hes Cells

Abstract hES cells provide an unique opportunity to study the earliest stages of hematopoietic commitment which are not easily accessible in the human embryo. To model early hematopoietic development, we cultured H1 and H9 hES cell lines on OP9 stromal cells without the addition of cytokines. On day 2 of co-culture, hES cells up-regulated brachyury expression and began to form mesodermal-like colonies. A transient population of blast colony-forming cells (CFCs) with the potential to differentiate into blood and endothelial cells was detected on days 3–6 of co-culture. CD34+ cells first appeared on day 3–4 of co-culture, which was coincident with induction of the transcription factors GATA-1, GATA-2 and SCL. CD43+ and CD41a+ cells along with CFCs emerged 2 days later within CD34+ population; 3–4 days before the appearance of CD45+ cells. We were able to obtain up to 20% of CD34+ cells from hES/OP9 co-culture and isolate up to 107 CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8–9 days of culture. The hES cell-derived CD34+ cells were highly enriched in CFCs, displayed CD90+CD117+CD164+CD38- phenotype of primitive hematopoietic progenitors, and contained ALDHhigh cells as well cells with verapamil-sensitive ability to efflux rhodamine 123. Isolated CD34+ cells differentiated into lymphoid (NK cells) as well as myeloid (neutrophils and macrophages) lineages when cultured on MS-5 stromal cells in the presence of SCF, Flt3-L, IL7 and IL3. These data indicate that hES cell/OP9 co-culture reproduces the major events that are observed during embryonal hematopoietic development, including the formation of lympho-myeloid progenitors. We employed OP9 system for identification of the phenotype of early hematopoietic progenitors in humans and to directly differentiate hES cells into different blood lineages.

scholarly journals Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures

Blood ◽  
2006 ◽  
Vol 109 (7) ◽  
pp. 2679-2687 ◽  
Author(s):  
Marion Kennedy ◽  
Sunita L. D'Souza ◽  
Macarena Lynch-Kattman ◽  
Staci Schwantz ◽  
Gordon Keller
Keyword(s):  
Endothelial Cells ◽  
Cell Differentiation ◽  
Receptor Tyrosine Kinase ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Vegf Receptor ◽  
Es Cell ◽  
Hematopoietic Development ◽  
Transient Population ◽  
First Time

Abstract The onset of hematopoiesis in the mouse embryo and in the embryonic stem (ES) cell differentiation model is defined by the emergence of the hemangioblast, a progenitor with both hematopoietic and vascular potential. While there is evidence for the existence of a hemangioblast in the mouse, it is unclear if this progenitor develops during the establishment of the human hematopoietic system. In this report, we have mapped hematopoietic development in human ES cell (hESC) differentiation cultures and demonstrated that a comparable hemangioblast population exists. The human hemangioblasts were identified by their capacity to generate blast colonies that display both hematopoietic and vascular potential. These colony-forming cells express the receptor tyrosine kinase KDR (VEGF receptor 2) and represent a transient population that develops in BMP-4–stimulated embryoid bodies (EBs) between 72 and 96 hours of differentiation, prior to the onset of the primitive erythroid program. Two distinct types of hemangioblasts were identified, those that give rise to primitive erythroid cells, macrophages, and endothelial cells and those that generate only the primitive erythroid population and endothelial cells. These findings demonstrate for the first time the existence of the human hemangioblast and in doing so identify the earliest stage of hematopoietic commitment.

Human Embryonic Stem Cells Demonstrate That Mpl Expression Initially Appears on a Population of CD31+ Endothelial Cells with Hemogenic Potential.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3617-3617
Author(s):  
Tamihiro Kamata ◽  
Yuping Gong ◽  
Shaohui Wang ◽  
Andrew D. Leavitt
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Cell Population ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Adherent Cells ◽  
Hematopoietic Development ◽  
Cd34 Cells ◽  
Early Human

Abstract Mammalian hematopoiesis occurs adjacent to endothelial cells in the yolk sac and in the AGM region during fetal development, and in vivo cell labeling with Ac-LDL in fetal birds and mice indicates that hematopoietic cells can arise from endothelial cells. Blood forming potential has also been reported in endothelial cells isolated from human fetal liver and fetal bone marrow. However, the role of endothelial cells in the generation of hematopoietic cells remains poorly understood. While thrombopoietin (TPO) and its receptor, Mpl, are known for their critical role in megakaryocytopoiesis, Mpl−/ − mice and patients with congenital amegakaryocytic thrombocytopenia demonstrate that Mpl signaling is also important for the establishment and/or maintenance of hematopoietic stem cells. However, the nature of that role and the relevant Mpl-expressing cells remain unknown. To determine if Mpl signaling in early hematopoiesis involves hemogenic endothelium, we used human embryonic stem cells (hESCs) co-cultured with OP9 stromal cells, a robust model system for generating human hematopoietic cells. We found that CD34+ cells appeared by day 3–4 of co-culture, followed one day later by the appearance of CD31+ cells, nearly all of which appeared within the CD34+ population. The CD34+ cells increased to 20–40% of the total cell population by day 10, with nearly half co-expressing CD31+. Mpl+ cells first appeared on day 6–7, almost exclusively within the CD31+ population, increasing to 5–10% of total cells by day 10. The hESC:OP9 system generated a vascular-like network lined with von Willebrand Factor-expressing cells, which combined with FACS data showing the onset of Mpl expression on CD31+ cells, suggested that Mpl may first be expressed on vascular lining cells. Interestingly, Mpl expression precedes the detection of CD45+ cells, which are first observed on day 8–9. Moreover, round CD45+ cells appeared within the vascular networks, raising the possibility that vascular lining cells may give rise to hematopoietic cells. To begin to characterize the role of TPO/Mpl signaling in the CD31+ cell population, FACS-sorted CD31+ cells from day-9 and day-10 cultures were plated onto a fibronectin (FN)-coated surface, and non-adherent cells were removed after 1 hour. The adherent CD31+ cells uptake Ac-LDL and express vWF, characteristic of endothelial cells. When cultured in media supplemented with endothelial growth factors, including VEGF, EGF, bFGF, IGF-1, and heparin, the FN-adherent cells generated areas of cobblestone-like cell clusters and CD45+/CD34+ cells. When cultured in the same conditions plus TPO, the number of cobblestone-like clusters increased, and the number of CD45+/CD34+ hematopoietic cells generated increased 3–4 fold. Our data demonstrate that Mpl is expressed on a CD31+, vWF-expressing cell population with hemogenic potential, and that TPO/Mpl signaling increases the yield of hematopoietic cells generated from these cells. Ongoing sorting experiments, including the isolation and characterization of CD31+/Mpl+ cells, will further our understanding of where and how Mpl signaling affects early human hematopoietic development. HESCs provide a novel system for defining the role of TPO/Mpl signaling in early human hematopoietic development, which may lead to improved treatment of hematopoietic disorders.

Human embryonic stem cell–derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 617-626 ◽  
Author(s):  
Maxim A. Vodyanik ◽  
Jack A. Bork ◽  
James A. Thomson ◽  
Igor I. Slukvin
Keyword(s):  
Stem Cell ◽  
Stromal Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Efficient Production ◽  
Alternative Source ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Hes Cells

AbstractEmbryonic stem (ES) cells have the potential to serve as an alternative source of hematopoietic precursors for transplantation and for the study of hematopoietic cell development. Using coculture of human ES (hES) cells with OP9 bone marrow stromal cells, we were able to obtain up to 20% of CD34+ cells and isolate up to 107 CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8 to 9 days of culture. The hES cell–derived CD34+ cells were highly enriched in colony-forming cells, cells expressing hematopoiesis-associated genes GATA-1, GATA-2, SCL/TAL1, and Flk-1, and retained clonogenic potential after in vitro expansion. CD34+ cells displayed the phenotype of primitive hematopoietic progenitors as defined by co-expression of CD90, CD117, and CD164, along with a lack of CD38 expression and contained aldehyde dehydrogenase–positive cells as well as cells with verapamil-sensitive ability to efflux rhodamine 123. When cultured on MS-5 stromal cells in the presence of stem cell factor, Flt3-L, interleukin 7 (IL-7), and IL-3, isolated CD34+ cells differentiated into lymphoid (B and natural killer cells) as well as myeloid (macrophages and granulocytes) lineages. These data indicate that CD34+ cells generated through hES/OP9 coculture display several features of definitive hematopoietic stem cells.

scholarly journals Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures

Blood ◽  
2006 ◽  
Vol 108 (6) ◽  
pp. 2095-2105 ◽  
Author(s):  
Maxim A. Vodyanik ◽  
James A. Thomson ◽  
Igor I. Slukvin
Keyword(s):  
Stromal Cells ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Surface Expression ◽  
Embryonic Stem Cell Differentiation ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Development ◽  
B Lymphoid ◽  
Definitive Hematopoiesis

AbstractDuring hematopoietic differentiation of human embryonic stem cells (hESCs), early hematopoietic progenitors arise along with endothelial cells within the CD34+ population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays, their phenotype has not been defined. Here, using hESC differentiation in coculture with OP9 stromal cells, we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45, persisted on differentiating hematopoietic cells, and reliably separated the hematopoietic CD34+ population from CD34+CD43–CD31+KDR+ endothelial and CD34+CD43–CD31–KDR– mesenchymal cells. Furthermore, we demonstrated that the first-appearing CD34+CD43+CD235a+CD41a+/–CD45– cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34+CD43+CD41a–CD235a–CD45– cells. These cells were negative for lineage-specific markers (Lin–), expressed KDR, VE-cadherin, and CD105 endothelial proteins, and expressed GATA-2, GATA-3, RUNX1, C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34+CD43+CD45–Lin– cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34+CD43+CD45+Lin– cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3highGATA3lowRUNX1lowPU1highMPOhighIL7RAhigh gene expression profile.

TET2 Favors Mesoderm and Hematopoietic Differentiation in Human Embryonic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2418-2418
Author(s):  
Barbara da Costa Reis Monte Mor ◽  
Thierry Langlois ◽  
Nathalie Droin ◽  
Elodie Pronier ◽  
Jean-Pierre Le Couédic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Conflicts Of Interest ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Development ◽  
Neuronal Genes ◽  
Hes Cells

Abstract Abstract 2418 TET2 belongs to the TET family proteins that catalyze 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (hmc) and plays an important role in normal and malignant adult hematopoiesis. The role of TET2 in human hematopoietic development remains unknown. Here we show that TET2 is expressed at low level in human embryonic stem (hES) cell lines and that its expression increases during hematopoietic differentiation in three different hES. TET2 knockdown does not modify hmc level and pluripotent properties of ES cells. However TET2 depletion by two different shRNA skewed differentiation into neuroectoderm at the expense of endoderm and mesoderm. This was associated with a decrease or an increase in promoter methylation of neuroectoderm and meso/endoderm genes, respectively during hES specification. Subsequently, we observed a decrease in hematopoietic progenitors (CD34+CD43+) and their cloning capacities due to a marked increase in apoptosis. Alteration of hematopoietic differentiation was coupled with a profound alteration in gene expression with up and down regulated genes including the abnormal expression of neuronal genes in hematopoietic cells. Thus our results suggest that TET2 regulates embryonic development by inhibiting neuroectoderm specification and enabling hematopoietic differentiation in hES cells. Disclosures: No relevant conflicts of interest to declare.

CD43 Defines Early Hematopoietic Progenitors Derived from Human Embryonic Stem Cells (hESC).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3216-3216
Author(s):  
Maxim A. Vodyanik ◽  
James A. Thomson ◽  
Igor I. Slukvin
Keyword(s):  
Endothelial Cells ◽  
Embryonic Stem ◽  
Hematopoietic Progenitors ◽  
Lineage Specification ◽  
Total Recovery ◽  
Cd34 Cells ◽  
Magnetic Sorting ◽  
Cell Fractions ◽  
High Level ◽  
Selection Of

Abstract We have demonstrated that co-culture of hESCs with OP9 bone-marrow stromal cells induced efficient hESC differentiation into hematopoietic colony-forming cells (CFCs). During hESC/OP9 co-culture, the first CFCs were detectable 1–2 days after emergence of CD34+ cells (day 4–5 of co-culture) and 3–4 days before generation of CD45+ cells, indicating that early CFCs arose in the CD45- population. To define phenotype of early hematopoietic progenitors, we examined the expression of CD41a, CD43, CD61 and glycophorin A (CD235a) on the CD34+ cells in the course of hESC/OP9 co-culture. The first detectable CD34+ cells expressed a high level of VEGF-R2 (KDR) and were CD41a, CD43, CD61 and CD235a negative. Appearance of CFCs was accompanied with down-regulation of KDR and almost simultaneous expression of CD41a, CD43 and CD235a on the CD34+ cells. CD43+ cells gradually increased thereafter, whereas the expression of CD41a was retained on the subset of CD43+ cells that co-express CD235a and CD61. CD45+ cells emerged within CD43+ population and the most of CD45+ cells did not express CD235a, CD41a or CD61. The expression of CD34 was decreased progressively on CD43+CD41a+CD235a+CD61+ cells, but retained by CD43+CD45+ cells. To assay hematopoietic potential of defined subsets, CD34+ cells were isolated by magnetic sorting and further separated into CD34+ CD43+ and CD34+CD43− cell fractions. In addition, CD34−CD43+ cells were isolated from CD34− fraction. CD34+ selection markedly enriched all types CFCs except of E-CFCs. All GEMM-CFCs were found within CD34+ population and none within CD34−. However, CD34− cell fraction contained E-CFCs and minimal numbers of M-CFCs and GM-CFCs. CD34+ cells were also heterogenous in morphologic appearance and contained endothelial cells that were capable to endocytose Ac-LDL. Positive selection of CD43+ cells resulted in total recovery of all types of CFCs. E-CFCs, which consistently detected in the CD34- fraction, were also recovered by CD43 selection. Morphologically, CD43+ cells comprised a homogenous blast-like population and were lack of endothelial-like cells. Thus, expression of CD43 molecule (leukosialin/sialophorin) is detected on the earliest clonogenic hematopoietic progenitors before expression of CD45, persists on developing hematopoietic cells after their lineage specification and most likely defines the divergence of hematopoiesis from endothelial cells during early development in humans.

Human Embryonic Stem Cell-Specific Micrornas Promote Full Programming of Induced Pluripotent Stem Cells Derived From CD34+ Cord Blood Cells Stored Frozen for More Than 20 Years.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2622-2622
Author(s):  
Man Ryul Lee ◽  
Nutan Prasain ◽  
Young-June Kim ◽  
Mervin C. Yoder ◽  
Hal E. Broxmeyer
Keyword(s):  
Transcription Factors ◽  
Cord Blood ◽  
Production Efficiency ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Self Renewal ◽  
Ips Cell ◽  
Cd34 Cells ◽  
Induced Pluripotent ◽  
Hes Cells

Abstract Abstract 2622 Numerous somatic cells can be reprogrammed by ectopic expression of defined transcription factors including OCT4, SOX2, MYC, KLF4 (Yamanaka's factors), NANOG, and LIN28 to pluripotent cells, referred to as induced pluripotent stem (iPS) cells which can then be differentiated into a variety of somatic cell types. Production efficiency of iPS cells by these transcription factors is extremely low, and therefore a large portion of the cells remain unprogrammed or incompletely reprogrammed. Thus, identification of additional factors required for enhancing iPS cells production efficiency has been an intensive research subject. We generated iPS cell from CD34+ cells of human umbilical cord blood that had been frozen in an unseparated state for twenty plus years using lentiviruses expressing Yamanaka's factors. During the iPS cell production processes, we monitored cell surface expression of TRA1-60, a marker for human embryonic stem (hES) cells, within colonies using a live cell staining method. Three to four weeks after gene transduction, TRA1-60 positive cells emerged in about 5% of the colonies. So, we mechanically separated TRA1-60 negative cells from TRA1-60 positive cells in colonies. Further culture did not allow TRA1-60 negative cells to convert to TRA1-60 positive cells even after 10 passages, indicating that TRA1-60 negative cells were stable at an incompletely reprogrammed state. TRA1-60 negative cells were similar to hES cells in morphology and still demonstrated expression of exogenous Yamanaka's factors. TRA1-60 negative cells were distinct from TRA1-60 positive cells with regards to methylation pattern of OCT4 promoter regions and differentiation potential. In contrast to TRA1-60 positive cells, incompletely reprogrammed TRA1-60 negative cells lacked hESC-specific miRNAs (miR-302 and 371 clusters), which are known to be involved in controlling self-renewal and pluripotency of hES cells. We hypothesized that these miRNAs can promote a transition from incompletely to fully reprogrammed iPS cells. To test this hypothesis, we introduced the miRNA clusters of miR-302 and 371 using lentiviruses to TRA1-60 negative incompletely reprogrammed cell to determine whether these miRNAs could convert TRA-60 negative cells to TRA1-60 positive completely reprogrammed cells. Our results showed that these miRNAs were able to convert more than 10% of TRA1-60 negative incompletely reprogrammed cells to TRA1-60 positive iPS cells with characteristics of completely reprogrammed iPS cells, such as differentiation of three germ layers and acquisition of typical hES cell-specific cell cycling patterns with an unrestricted G1 to S phase transition. These results indicate that hES-specific miRNAs have a strong potential to promote partially reprogrammed cord blood CD34+ cells to iPS cells with extensive self-renewal capacity. Our study suggests that current techniques with low iPS cell production efficiency can be improved by ectopic overexpression of hES-specific miRNAs (miR-302 and371 clusters). Disclosures: Broxmeyer: Corduse: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Notch1 acts via Foxc2 to promote definitive hematopoiesis via effects on hemogenic endothelium

Blood ◽  
2015 ◽  
Vol 125 (9) ◽  
pp. 1418-1426 ◽  
Author(s):  
Il Ho Jang ◽  
Yi-Fen Lu ◽  
Long Zhao ◽  
Pamela L. Wenzel ◽  
Tsutomu Kume ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation ◽  
Zebrafish Embryos ◽  
Hemogenic Endothelium ◽  
Key Points ◽  
Definitive Hematopoiesis

Key Points Notch1 induction promotes specification of hemogenic endothelial cells during embryonic stem cell differentiation. Foxc2 functions downstream of Notch in specification of hemogenic endothelium in mouse and zebrafish embryos.

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