Developmental Potentials of Human Embryonic Stem Cells Lacking PIG-A and GPI-Anchored Proteins.

Abstract Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal disorder of hematopoietic stem cells (HSCs) acquiring mutations in the PIG-A gene. Its product (PIG-A protein) is required for biosynthesis of dozens of cell surface proteins to be linked to glycosyl-phosphatidyl-inositol (GPI) molecule and anchored on plasma membrane. The underlying mechanisms of PIG-A mutant clonal dominance in PNH patients and the close relationship of PNH to other bone marrow failure diseases (aplastic anemia and myelodysplasia syndrome) and leukemia remain unknown. Establishing a mutation of PIG-A gene in human HSCs from healthy donors remains unfeasible due to the current inability to expand and select clonal HSCs in culture. Although mouse models have been generated by disrupting conditionally the mouse Pig-a gene, the existing Pig-a null mice indeed lacking GPI-APs in blood cells did not replicate faithfully PNH pathological symptoms. To create a human cell-based, prospective experimental system to investigate the effects of PIG-A/GPI-AP deficiency and pathophysiology of PNH, we made mutated hES cells lacking GPI-APs. FACS analysis revealed that two independent hES clones lack cell-surface expression of CD55 and CD59, as well as CD90/Thy and Cripto that are preferentially expressed in undifferentiated hES cells. However, the cell-surface expression of these GPI-APs can be restored by a lentiviral vector inducibly expressing the PIG-A cDNA. Like mouse ES cells, lack of PIG-A/GPI-APs did not affect the growth of undifferentiated hES cells in culture. Unlike mouse ES cells, however, PIG-A/GPI-AP deficient hES cells formed embryoid bodies normally in culture. RT-PCR analysis of marker gene expression indicated that commitment to the 3 (somatic) germ layers appeared normal within embryonic bodies from either the mutated or wildtype hES cells. However, formation of extra-embryonic cells such as trophoblasts from the PIG-A/GPI-AP deficient hES cells is defective in both embryoid body formation and BMP4-induced assays. Expression of trophoectoderm-specific genes such as hCGalpha could not be induced in PIG-A/GPI-AP deficient hES cells upon BMP4 induced trophoectodermal differentiation. The induction of other trophoectoderm markers such as hCGbeta and CDX2 was also significantly reduced after the BMP4 treatment. The lack of tropho-ectoderm was further confirmed by lacking of hCG hormone production. The defect in trophoectoderm differentiation from the PIG-A/GPI-AP deficient hES cells was restored by the expression of the PIG-A cDNA in the mutated hES cells. For somatic cell differentiation, we are currently examining the effects of PIG-A/GPI-AP deficiency beyond the initial differentiation commitment during embryonic body formation stage. Along mesodermal and hematopoietic differentiation, we found that the PIG-A/GPI-AP deficiency in hES cells had little effect on the formation of CD34+ cells, a precursor cell population for human hematopoietic and endothelial lineages. We are currently examining the effects of PIG-A/GPI-AP deficiency on properties of hematopoietic cells derived from the mutated and normal hES cells. This study represents one of first cases that hES cells may provide a prospective research model to investigate genetic and developmental basis of human diseases.

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

Blood ◽

10.1182/blood.v87.7.2740.bloodjournal8772740 ◽

1996 ◽

Vol 87 (7) ◽

pp. 2740-2749 ◽

Cited By ~ 83

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.

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Separate Developmental Programs for HLA-A and -B Cell Surface Expression during Differentiation from Embryonic Stem Cells to Lymphocytes, Adipocytes and Osteoblasts

PLoS ONE ◽

10.1371/journal.pone.0054366 ◽

2013 ◽

Vol 8 (1) ◽

pp. e54366 ◽

Cited By ~ 4

Author(s):

Hardee J. Sabir ◽

Jan O. Nehlin ◽

Diyako Qanie ◽

Linda Harkness ◽

Tatyana A. Prokhorova ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Surface ◽

B Cell ◽

Embryonic Stem ◽

Surface Expression ◽

Cell Surface Expression ◽

Developmental Programs

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The generation and use of human embryonic stem cells

Oxford Textbook of Endocrinology and Diabetes ◽

10.1093/med/9780199235292.003.0805 ◽

2011 ◽

pp. 1173-1179

Author(s):

Mikael C. O. Englund ◽

Christopher L. R. Barratt

Keyword(s):

Parkinson’S Disease ◽

Stem Cells ◽

Parkinson's Disease ◽

Drug Discovery ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Es Cells ◽

Embryonic Stem ◽

Mouse Es Cells ◽

Hes Cells

Ever since the first human embryonic stem cells (hES) were successfully derived and propagated in 1998 (1), an obvious topic of discussion has been the development of novel therapies based on stem cell technology for a number of diseases and conditions. Targets could include type 1 diabetes, Alzheimer’s disease, spinal cord injury, and Parkinson’s disease to name a few. hES cells can also be used for tissue engineering, to replace for example bone and cartilage, and for drug discovery. Exciting proof of principal experiments in animals demonstrate the clinical potential in this field. For example, in a rat model of Parkinson’s disease, dopamine neural grafts derived from mouse Es cells showed long-term survival, the production of dopamine and, importantly, persistent improvements in movement behaviour (2). The promises of these potential treatments is enormous. However, there are many hurdles to overcome before a therapy based on stem cells is a clinical reality. We outline (A) the variety of methods to derive hES cells including somatic cell nuclear transfer (SCNT) and describe the challenges and possible avenues of further use; (B) discuss the development of clinical grade hES cells and their use in the drug discovery process; and (C) alternative strategies to patient specific therapy including induced adult pluripotent stem cells (iPS cells).

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Impaired Cell Surface Expression of HLA-B Antigens on Mesenchymal Stem Cells and Muscle Cell Progenitors

PLoS ONE ◽

10.1371/journal.pone.0010900 ◽

2010 ◽

Vol 5 (5) ◽

pp. e10900 ◽

Cited By ~ 13

Author(s):

Adiba Isa ◽

Jan O. Nehlin ◽

Hardee J. Sabir ◽

Tom E. Andersen ◽

Michael Gaster ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Surface ◽

Muscle Cell ◽

Surface Expression ◽

Cell Surface Expression

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The cytoplasmic domain of Mpl receptor transduces exclusive signals in embryonic and fetal hematopoietic cells

Blood ◽

10.1182/blood.v100.6.2063.h81802002063_2063_2070 ◽

2002 ◽

Vol 100 (6) ◽

pp. 2063-2070

Author(s):

Cécile Challier ◽

Laurence Cocault ◽

Rolande Berthier ◽

Nadine Binart ◽

Isabelle Dusanter-Fourt ◽

...

Keyword(s):

Stem Cells ◽

Fetal Liver ◽

Es Cells ◽

Cytoplasmic Domain ◽

Embryoid Bodies ◽

Chimeric Receptor ◽

Hematopoietic Differentiation ◽

Es Cell ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem

The Mpl receptor plays an important role at the level of adult hematopoietic stem cells, but little is known of its function in embryonic and fetal hematopoiesis. We investigated the signals sent by the MPL cytoplasmic domain in fetal liver hematopoietic progenitors and during embryonic stem (ES) cell hematopoietic commitment. Mpl was found to be expressed only from day 6 of ES cell differentiation into embryoid bodies. Therefore, we expressed Mpl in undifferentiated ES cells or in fetal progenitors and studied the effects on hematopoietic differentiation. To avoid the inadvertent effect of thrombopoietin, we used a chimeric receptor, PM-R, composed of the extracellular domain of the prolactin receptor (PRL-R) and the transmembrane and cytoplasmic domains of Mpl. This allowed activation of the receptor with a hormone that is not involved in hematopoietic differentiation and assessment of the specificity of responses to Mpl by comparing PM-R with another PRL-R chimeric receptor that includes the cytoplasmic domain of the erythropoietin receptor (EPO-R) ([PE-R]). We have shown that the cytoplasmic domain of the Mpl receptor transduces exclusive signals in fetal liver hematopoietic progenitors as compared with that of EPO-R and that it promotes hematopoietic commitment of ES cells. Our findings demonstrate for the first time the specific role of Mpl in early embryonic or fetal hematopoietic progenitors and stem cells.

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YY1 Promotes SCF/c-Kit Signaling and HSC Self-Renewal in Fetal Hematopoiesis

Blood ◽

10.1182/blood-2020-137254 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-27

Author(s):

Deependra Kumar Singh ◽

Zhanping Lu ◽

Shuai Jia ◽

Xiaona You ◽

Jing Zhang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Surface ◽

Fetal Liver ◽

Surface Expression ◽

Cell Surface Expression ◽

Self Renewal ◽

Hematopoietic Stem ◽

Phosphorylated Akt ◽

Fetal Hematopoiesis

Fetal hematopoietic stem cells (HSCs) exhibit markedly different properties as compared to adult HSCs including cell surface marker expression, proliferation state and repopulation capacity. Changes of HSC activity in postnatal mice is defined by a process of decreasing in cell cycle and entering into quiescence. Yin Yang 1 (YY1) is a ubiquitous transcription factor and mammalian Polycomb Group (PcG) Protein with important functions to regulate embryonic development, lineage differentiation and cell proliferation. While homozygote deletion of YY1 in mice results in lethality at the peri-implantation stage, heterozygote deletion of YY1 causes severe developmental defects. By conditionally deleting YY1 in adult hematopoietic system, our previous study showed that YY1 is an essential regulator for adult hematopoiesis by promoting HSC long-term self-renewal and maintaining adult HSC quiescence. In contrast to adult HSCs, in which quiescence is a fundamental characteristic, over 95% of fetal HSCs are in an active cell cycle to rapidly generate homeostatic levels of blood cells for oxygen transport and immune system development in the growing organism. Herein, we assessed whether YY1 was also required for maintaining fetal HSC pool and regulating fetal HSC functions, and what was the underlying mechanism by which YY1 regulated fetal HSCs. To test how loss-of-function of YY1 impacted fetal hematopoiesis, Yy1f/f mice in which the Yy1 promoter region and exon 1 are flanked by loxP sites, were crossed to Vav-Cre mice to generate heterozygous Yy1f/+ Vav-cre mice. The Vav promoter drives Cre recombinase expression specifically in fetal liver hematopoietic cell starting at day E11.5. Yy1f/+ Vav-cre mice were then subsequently bred with Yy1f/f mice to generate homozygous Yy1f/f Vav-cre mice. Among 141 pups resulting from breeding Yy1f/fto Yy1f/+ Vav-Cre, only 7 were Yy1f/f Vav-Cre (n=7) and was significantly lower than the estimated number (n=35) according to the Mendelian ratio (P<0.05). All Yy1f/f Vav-Cre pups died within 72 hours after birth, which supported essential role of YY1 in fetal hematopoiesis and survival. At E14.5 of fetal development, Yy1f/f Vav-Cre fetuses had reduced numbers of hematopoietic stem and progenitor cells in the liver. In addition, YY1 deficient fetal HSCs failed to self-renew in primary and secondary bone marrow transplantation assays. Colony formation assay showed that fetal liver cells from Yy1f/f Vav-Cre mice failed to form CFU-GEMM, CFU-GU and BFU-E compared to Vav-Cre control. While YY1 promotes SCF/c-Kit signaling in adult HSCs, it does not impact c-Kit cell surface expression in early T cell progenitors (unpublished data). To assess YY1 impact on SCF/c-Kit axle in fetal HSCs, c-Kit transcript level, c-Kit median fluorescence intensity and phosphorylated AKT were measured. Similar as its function in adult HSCs, YY1 deficient fetal HSCs had decreased Kit transcript expression, decreased c-Kit cell surface expression and decreased SCF/c-Kit signaling. Our results supported that YY1 is required for maintaining a continuous pool of HSCs in fetal liver and is critical for fetal HSC long-term self-renewal and differentiation. Similar as its function in adult HSCs, YY1 promotes SCF/c-Kit signaling in fetal HSCs. Disclosures No relevant conflicts of interest to declare.

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The cytoplasmic domain of Mpl receptor transduces exclusive signals in embryonic and fetal hematopoietic cells

Blood ◽

10.1182/blood.v100.6.2063 ◽

2002 ◽

Vol 100 (6) ◽

pp. 2063-2070 ◽

Cited By ~ 2

Author(s):

Cécile Challier ◽

Laurence Cocault ◽

Rolande Berthier ◽

Nadine Binart ◽

Isabelle Dusanter-Fourt ◽

...

Keyword(s):

Stem Cells ◽

Fetal Liver ◽

Es Cells ◽

Cytoplasmic Domain ◽

Embryoid Bodies ◽

Chimeric Receptor ◽

Hematopoietic Differentiation ◽

Es Cell ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem

Abstract The Mpl receptor plays an important role at the level of adult hematopoietic stem cells, but little is known of its function in embryonic and fetal hematopoiesis. We investigated the signals sent by the MPL cytoplasmic domain in fetal liver hematopoietic progenitors and during embryonic stem (ES) cell hematopoietic commitment. Mpl was found to be expressed only from day 6 of ES cell differentiation into embryoid bodies. Therefore, we expressed Mpl in undifferentiated ES cells or in fetal progenitors and studied the effects on hematopoietic differentiation. To avoid the inadvertent effect of thrombopoietin, we used a chimeric receptor, PM-R, composed of the extracellular domain of the prolactin receptor (PRL-R) and the transmembrane and cytoplasmic domains of Mpl. This allowed activation of the receptor with a hormone that is not involved in hematopoietic differentiation and assessment of the specificity of responses to Mpl by comparing PM-R with another PRL-R chimeric receptor that includes the cytoplasmic domain of the erythropoietin receptor (EPO-R) ([PE-R]). We have shown that the cytoplasmic domain of the Mpl receptor transduces exclusive signals in fetal liver hematopoietic progenitors as compared with that of EPO-R and that it promotes hematopoietic commitment of ES cells. Our findings demonstrate for the first time the specific role of Mpl in early embryonic or fetal hematopoietic progenitors and stem cells.

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Effect of Imatinib on Proliferation, Apoptosis and Expression of Platelet Derived Growth Factor Receptor By Mesenchymal Stem Cells

Blood ◽

10.1182/blood.v124.21.5135.5135 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5135-5135

Author(s):

Fatima Aerts-Kaya ◽

Gulen Guney ◽

Sule Unal ◽

Duygu Uckan-Cetinkaya

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Surface ◽

Osteogenic Differentiation ◽

Growth Factor Receptor ◽

Annexin V ◽

Surface Expression ◽

Platelet Derived Growth Factor ◽

Cell Surface Expression

Abstract Introduction Imatinib Mesylate (IM) is a tyrosine kinase inhibitor (TKI), which targets Platelet Derived Growth Factor Receptor (PDGF-R), c-kit and BCR-ABL and is used in the treatment of Gastrointestinal Stromal Tumors (GIST) and Chronic Myeloid Leukemia (CML). Patients using Imatinib for a long time display abnormalities in bone metabolism (Vandyke et al. J Clin Endocrinol Metab 2013). In vitro, IM affects osteoblastic differentiation through inhibition of PDGF/PDGF-Rb signaling, as well as osteoclast function/differentiation through modulation of PDGF/PDGF-Ra signaling (Berman et al. Leuk Res 2013). Conflicting reports have shown differential effects on in vivo bone marrow density. Confounding factors include patient age (pediatric ve adult), type of bone (osteochondral vs trabecular), duration and dose of IM treatment. Mesenchymal Stem Cells (MSCs) are cells with great regenerative potential and differentiate into adipogenic, osteogenic cells and multiple other cell lineages (Bianco et al. Cell Stem Cell 2008). MSCs express high cell surface levels of PDGF-Rb and intermediate levels of PDGF-Ra and are therefore likely to be affected by IM treatment. Here, we wanted to assess the effect of IM on proliferation and apoptosis of MSCs as well as on surface expression of PDGF-Rb and PDGF-Ra. Methods Healthy human bone marrow MSCs were isolated using Ficoll and plastic adherence and cultured up till passage 3. Proliferation assays were performed using Real Time Cell Analysis (XCELLigence, Roche) to determine optimal in vitro doses of IM (Novartis). IM was used at doses from 2,5 uM to 20 uM and found to be optimal at 5 uM. PDGF-Rb (CD140b) and PDGF-Ra (CD140a) surface expression were measured using monoclonal antibodies and read using a FACSARIA (Becton Dickinson). Apoptosis was assessed using Annexin-V and Propidium Iodide. Adipogenic and osteogenic differentiation was evaluated after 21 days in differentiation media with or without IM, using spectrophotometric quantitation of levels of Oil Red O (Adipogenic differentiation) and Calcium Phosphate (Osteogenic differentiation). Results IM inhibited proliferation of MSCs in a dose-dependent fashion, with doses > 5 uM resulting in severe suppression of proliferation. Inhibition of proliferation by IM could be overcome by increasing cell densities of MSCs, but not by addition of PDGF-BB. Co-treatment with IM and PDGF-BB resulted in more pronounced suppression of MSC proliferation. Treatment with IM resulted in a decrease of cell surface expression of both PDGF-Rb from 97,4±2,36% to 77,3±0,13% (n=3, p<0.02) and PDGF-Ra from 18,4% to 6,4% (n=1). Addition of PDGF-BB resulted in a further decrease in cell surface expression of PDGF-Rb, but had no effect on expression of PDGF-Ra. IM increased apoptosis levels (Annexin-V positive cells) about twofold. Addition of 5 or 10 ng/mL PDGF could completely abrogate this effect. Treatment of K562, a BCR-ABL positive CML cell line, with 5 uM IM suppressed proliferation of K562 four-fold, but had no obvious effect on levels of apoptosis. Treatment of MSCs with IM during differentiation revealed no clear effect on adipogenesis, but did increase osteogenic differentiation, as measured as an increase in Calcium-Phosphate. Conclusions Imatinib Mesylate inhibits PDGF/PDGF-R signaling through interference with tyrosine kinases. Here, we found that 5 uM IM not only affects MSC proliferation through inhibition of PDGF-receptor signaling, but also through downregulation of PDGF-Rb and PDGF-Ra cell surface expression. This dose is very close to the maximal plasma concentration of 4.6 uM observed in patients (Druker et al. New England J Med 2001). Addition of PDGF-BB enhanced the effects of IM on suppression of MSC proliferation, likely through further downregulation of surface PDGF-Rb expression, thus decreasing PDGF/PDGF-R signaling. PDGF signaling has been implicated in regulation of invasiveness of cancers and TKI have been used in the treatment of several types of cancer. Whether or not combination treatment of IM with PDGF-BB similarly affects proliferation/invasiveness of BCR-ABL+ CML cells, and PDGF-Ra positive GIST cells, remains to be investigated. Prolonged treatment with IM in patients has been shown to affect bone remodeling and bone densities. Our current results suggest that treatment with IM impacts BM-resident MSCs, supporting increased osteogenesis. Disclosures No relevant conflicts of interest to declare.

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The Hematopoietic Stem Cells Could Be Switched From Leukemic Cell Lines

Blood ◽

10.1182/blood.v122.21.4845.4845 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4845-4845

Author(s):

Jianda Hu ◽

Ting Yang ◽

Liangfang Zhu ◽

Xiaofeng Luo ◽

Xiaohong Yuan ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Surface ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Embryonic Stem ◽

Restriction Enzyme Digestion ◽

Hematopoietic Stem ◽

Primary Mouse ◽

Hes Cells

Abstract Background The hematopoietic potential from induced pluripotent stem cells (iPS) has been explored for some years. However, the direct reprogramming from the blood cells to hematopoietic progenitors bypass the pluripotency is even more attractive for the clinical practice. Methods The lentiviral vector encoding human reprogramming factors (Oct-4, Sox2, c-Myc, Klf4FOSCK) were transfected along with packaging plasmid pCMV-dR8.91 and envelope plasmid pCMV-VSV-G in 293T package cells to produce lentiviral particles. The construction was then confirmed by RT-PCR and restriction enzyme digestion anaysis, and the lentivirus titer was determined using GFP/DAPI based on cell count by Image J software. Primary mouse embryo fibroblast (PMEF) feeder cells were isolated and prepared from CF-1 inbred mouse strain to help maintain pluripotency and to provide a cellular matrix for stem cells growing. Lastly, the lentivirus carrying OSCK factors was used totransduce HL-60 cells in three consecutive rounds of spin-infection on the prepared feeder layers with an interval of 12 hours, while the lentivirus carrying GFP served as a negative control. The cell clusters were picked based on morphology and alkaline phosphatase(AP) staining. The stem cell properties were tested by the expression of cell surface antigens using Flow cytometry. and the mRNA expression of OCT4, SOX2, C-MYC,KLF4,etc. by QT-PCR. Results The lentiviral particles were successful packaged and used to infect HL-60 cells. We occasionally observed some Human embryonic stem cells (hES) cell-like colonies in between the cells around 14 days after infection. These cell colonies also showed similarity to hES cells in feeder dependency. Detection of cell surface CD34 by FCM showed that HL-60 cells were switched to be a hematopoietic fate, expression of CD34 from 1.77% to 98.42%. Simultaneously, expressions of the myeloid antigen CD13, CD117 decreased. The gene expressions of OCT4, SOX2 indicated that the exogenous gene were down-regulation or silence while the endogenous gene were up-regulation. However, the cell colonies can survive only for a short time which might due to the absence of the survival factors they require or the first hematopoietic microenvironment, like the yolk sac. Conclusion After being reprogrammed, the HL-60 cell derived colonies showed the similarity to hES cells in morphology, feeder dependency and the expression of stem cell antigens. However, they may need an appropriate microenvironment at different time points to be programmed into primitive hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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