Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 2003-2013 ◽  
Author(s):  
Maria Teresa Mitjavila-Garcia ◽  
Michel Cailleret ◽  
Isabelle Godin ◽  
Maria Manuela Nogueira ◽  
Karine Cohen-Solal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor

In this study, we have characterized the early steps of hematopoiesis during embryonic stem cell differentiation. The immunophenotype of hematopoietic progenitor cells derived from murine embryonic stem cells was determined using a panel of monoclonal antibodies specific for hematopoietic differentiation antigens. Surprisingly, the CD41 antigen (αIIb integrin, platelet GPIIb), essentially considered to be restricted to megakaryocytes, was found on a large proportion of cells within embryoid bodies although very few megakaryocytes were detected. In clonogenic assays, more than 80% of all progenitors (megakaryocytic, granulo-macrophagic, erythroid and pluripotent) derived from embryoid bodies expressed the CD41 antigen. CD41 was the most reliable marker of early steps of hematopoiesis. However, CD41 remained a differentiation marker because some CD41– cells from embryoid bodies converted to CD41+ hematopoietic progenitors, whereas the inverse switch was not observed. Immunoprecipitation and western blot analysis confirmed that CD41 was present in cells from embryoid bodies associated with CD61 (β3 integrin, platelet GPIIIa) in a complex. Analysis of CD41 expression during ontogeny revealed that most yolk sac and aorta-gonad-mesonephros hematopoietic progenitor cells were also CD41+, whereas only a minority of bone marrow and fetal liver hematopoietic progenitors expressed this antigen. Differences in CD34 expression were also observed: hematopoietic progenitor cells from embryoid bodies, yolk sac and aorta-gonad-mesonephros displayed variable levels of CD34, whereas more than 90% of fetal liver and bone marrow progenitor cells were CD34+. Thus, these results demonstrate that expression of CD41 is associated with early stages of hematopoiesis and is highly regulated during hematopoietic development. Further studies concerning the adhesive properties of hematopoietic cells are required to assess the biological significance of these developmental changes.

scholarly journals Co-Acquisition of RUNX1 and CSF3R Mutations Transforms Hematopoietic Progenitor Cells of CN Patients into More Primitive Highly Proliferative Blasts: Evidence in CN Patients and in a Mouse Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 223-223 ◽  
Author(s):  
Olga Klimenkova ◽  
Maksim Klimiankou ◽  
Amy Schmidt ◽  
Carol Stocking ◽  
Cornelia Zeidler ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
High Frequency ◽  
Expression Profiles ◽  
Hematopoietic Cells ◽  
Hematopoietic Progenitor Cells ◽  
Myeloid Differentiation ◽  
Hematopoietic Progenitors ◽  
Granulocytic Differentiation ◽  
Hematopoietic Progenitor

Abstract Severe congenital neutropenia (CN) is a preleukemic bone marrow failure syndrome with a high risk of evolving into leukemia or myelodysplastic syndrome (MDS). Recently we demonstrated a very high frequency of cooperating RUNX1 and CSF3R mutations in CN patients who developed leukemia or MDS (Skokowa, et al. Blood 2014). We proposed a novel molecular pathway of leukemogenesis: mutations in the cytokine receptor (G-CSFR) in combination with the second mutations in the hematopoietic transcription fator (RUNX1). In the majority of CN patients, CSF3R mutations were acquired prior to RUNX1 mutations. CSF3R mutations alone are unable to induce leukemia in CN patients or in mice expressing a transgenic d715 G-CSFR. Co-acquisition of RUNX1 mutations is an essential step in the leukemogenic transformation in CN. To characterize the expression signature of hematopoietic cells of CN/AML patients carrying CSF3R mutations prior to and after acquisition of RUNX1 mutations, we analyzed expression profiles of CD34+ hematopoietic cells of CN patient who developed AML. This patient acquired CSF3R mutation (p. Q718*) five years and RUNX1 mutation (p. R139G) 16 months prior to leukemia. We compared expression profiles of CD34+ cells harbouring CSF3R mutation only, or both CSF3R and RUNX1 mutations. Co-acquisition of RUNX1 and CSF3R mutations led to marked reduction of the expression of hematopoietic growth factors such as IL6 and NAMPT, inhibitors of cytokine signaling SOCS3, as well as of components of neutrophil granules OLFM4, DEFA4, MMP8, SLPI, CRISP3 and CTSG. At the same time expression levels of pro-proliferative downstream effectors of G-CSF such as STAT5A, STAT5B, SMAD1 and cyclin A1 (CCNA1) were dramatically elevated. Moreover, genes overexpressed in early hematopoietic stem/progenitor cells (HSPCs) as compared to more mature progenitors, such as DNTT, BAALC, CD109, HPGDS, PDLIM1, MLLT11 and FLT3 were strongly upregulated in CN/AML blasts harbouring both RUNX1 and CSF3R mutations. Intriguingly, elevated expression of DNTT, BAALC, CD109 and FLT3 was described previously in RUNX1-mutated de novo AML blasts (Mendler et al., JCO 2012). This genetic signature suggests rapid transformation of hematopoietic progenitors carrying mutated CSF3R into more primitive hematopoietic progenitors after acquisition of RUNX1mutation. To elucidate the role of cooperative CSF3R and RUNX1 mutations on the clonogenic capacity and myeloid differentiation of hematopoietic progenitors, we performed functional studies in mice. We transduced lineage negative (lin-) bone marrow hematopoietic progenitor cells of WT or transgenic d715 G-CSFR mice with lentiviral expression constructs containing either WT or mutated forms of RUNX1 cDNA. We used two different mutants of RUNX1 by introduction of mutations at amino acid positions 139 and 174. Acquired RUNX1 mutations in these amino acids were presented with high frequency in our cohort of CN/AML patients and in most of the cases were associated with acquired CSF3R mutations. We found that similar to the effect of CSF3R mutations, lin-hematopoietic cells of WT mice transduced with mutated RUNX1 alone did not show elevated clonogenic capacity in replating experiments. Interestingly, transduction of WT cells with RUNX1 mutants resulted in severely reduced numbers of CFU-G colonies but unaffected CFU-M and BFU-E colonies. Intriguingly, transduction of lin- hematopoietic cells from transgenic d715 G-CSFR mice with RUNX1 mutants resulted in a markedly elevated clonogenic capacity in replating experiments, as compared to cells transduced with WT RUNX1 or control vector: numbers of colonies after second replating were 7 and 8 times higher in RUNX1-R139G and RUNX1-R174X mutants, respectively, in comparison to RUNX1 WT transduced cells. Moreover, granulocytic differentiation of lin- cells from d715 G-CSFR mice transduced with RUNX1-R139G mutant was severely diminished, in comparison to cells transduced with WT RUNX1, as revealed by 5-fold reduction of CFU-G colonies. Taken together, co-acquisition of RUNX1 and CSF3R mutations shifted the hematopoietic differentiation program towards more primitive hematopoietic progenitors with elevated proliferative capacity and reduced myeloid differentiation, which ultimately lead to leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals A novel 37-Kd adhesive membrane protein from cloned murine bone marrow stromal cells and cloned murine hematopoietic progenitor cells

Blood ◽  
1993 ◽  
Vol 82 (5) ◽  
pp. 1436-1444 ◽  
Author(s):  
Y Shiota ◽  
JG Wilson ◽  
K Harjes ◽  
ED Zanjani ◽  
M Tavassoli
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Marrow Stromal Cells ◽  
Hematopoietic Progenitor Cells ◽  
Single Chain ◽  
Hematopoietic Progenitor

Abstract The adhesion of hematopoietic progenitor cells to bone marrow stromal cells is critical to hematopoiesis and involves multiple effector molecules. Stromal cell molecules that participate in this interaction were sought by analyzing the detergent-soluble membrane proteins of GBI/6 stromal cells that could be adsorbed by intact FDCP-1 progenitor cells. A single-chain protein from GBI/6 cells having an apparent molecular weight of 37 Kd was selectively adsorbed by FDCP-1 cells. This protein, designated p37, could be surface-radiolabeled and thus appeared to be exposed on the cell membrane. An apparently identical 37- Kd protein was expressed by three stromal cell lines, by Swiss 3T3 fibroblastic cells, and by FDCP-1 and FDCP-2 progenitor cells. p37 was selectively adsorbed from membrane lysates by a variety of murine hematopoietic cells, including erythrocytes, but not by human erythrocytes. Binding of p37 to cells was calcium-dependent, and was not affected by inhibitors of the hematopoietic homing receptor or the cell-binding or heparin-binding functions of fibronectin. It is proposed that p37 may be a novel adhesive molecule expressed on the surface of a variety of hematopoietic cells that could participate in both homotypic and heterotypic interactions of stromal and progenitor cells.

scholarly journals Murine yolk sac endoderm- and mesoderm-derived cell lines support in vitro growth and differentiation of hematopoietic cells

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2436-2443 ◽  
Author(s):  
MC Yoder ◽  
VE Papaioannou ◽  
PP Breitfeld ◽  
DA Williams
Keyword(s):  
Progenitor Cells ◽  
Cell Lines ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Yolk Sac ◽  
Hematopoietic Progenitor Cells ◽  
Visceral Endoderm ◽  
Hematopoietic Progenitor

Abstract The mechanisms involved in the induction of yolk sac mesoderm into blood islands and the role of visceral endoderm and mesoderm cells in regulating the restricted differentiation and proliferation of hematopoietic cells in the yolk sac remain largely unexplored. To better define the role of murine yolk sac microenvironment cells in supporting hematopoiesis, we established cell lines from day-9.5 gestation murine yolk sac visceral endoderm and mesoderm layers using a recombinant retrovirus vector containing Simian virus 40 large T- antigen cDNA. Obtained immortalized cell lines expressed morphologic and biosynthetic features characteristic of endoderm and mesoderm cells from freshly isolated yolk sacs. Similar to the differentiation of blood island hematopoietic cells in situ, differentiation of hematopoietic progenitor cells in vitro into neutrophils was restricted and macrophage production increased when bone marrow (BM) progenitor cells were cultured in direct contact with immortalized yolk sac cell lines as compared with culture on adult BM stromal cell lines. Yolk sac- derived cell lines also significantly stimulated the proliferation of hematopoietic progenitor cells compared with the adult BM stromal cell lines. Thus, yolk sac endoderm- and mesoderm-derived cells, expressing many features of normal yolk sac cells, alter the growth and differentiation of hematopoietic progenitor cells. These cells will prove useful in examining the cellular interactions between yolk sac endoderm and mesoderm involved in early hematopoietic stem cell proliferation and differentiation.

FDA-Approved Pharmacological Agents, Romiplostium and Oprelvekin, Synergistically Promote Megakaryocytic Differentiation From Human iPSCs In a Chemically Defined System

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1208-1208
Author(s):  
Yanfeng Liu ◽  
Yongxing Gao ◽  
Sid Shah ◽  
Lewis Becker ◽  
Linzhao Cheng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitor Cells ◽  
Patient Specific ◽  
Hematopoietic Progenitor ◽  
Free System ◽  
Serum Free ◽  
Megakaryocytic Differentiation

Abstract Platelets, anucleate cells derived from megakaryocytes (MKs) that are generated within the bone marrow, play an important role in the process of physiological hemostasis and in vascular repair. Low platelets in the blood stream result in bleeding risk in thrombocytopenic patients with liver failure, leukemia, or undergoing chemotherapy. Platelet transfusions remain the mainstay of treatment and require a constant supply of platelets. Because platelets from donor blood have a short life-span (only few days in storage), platelets are always in a short supply. In vitro generation of MKs and platelets from human induced pluripotent stem cells (hiPSCs) would provide a patient-specific renewable cell source of MKs and platelets to treat thrombocytopenic patients at risk of hemorrhage. We derived integration-free hiPSCs from peripheral blood cells of more than 20 individuals, and examined two methods of in vitro differentiation into MKs: i) co-culture on 10T1/2 cells or OP9 cells first developed by Takayama et al. (2010), and ii) a feeder-free and serum-free system by first forming embryoid bodies (EBs) in a chemically defined condition, similar to the recently published method of Pick et al. (2013). Although both methods gave rise with similar efficiency to CD41a+CD42a+ MKs with large cell size and high-ploidy DNA, we chose to focus on the feeder-free system that began with EB formation with centrifugal aggregation of hiPSCs (spin-EBs) because it is cheaper, faster, easier to scale up, and represents a chemically defined system. To investigate the effect of growth factors on hiPSC differentiation to MKs, we modified the spin-EB system to three steps: i) mesoderm induction and hematopoietic commitment in the presence of BMP4, VEGF, bFGF and SCF (day 0 to day 11), ii) hematopoietic progenitor and MK differentiation by adding TPO (day 11 to 14), and iii) MK maturation (day 14 to 19). To assess whether the FDA-approved pharmacological agent, Romiplostium (Nplate®, TPO analog), has a similar effect to TPO on MK differentiation from hiPSCs, we isolated hematopoietic progenitor cells at day 14, and differentiated them into MKs with Romiplostium or TPO. Our data demonstrated that Romiplostium (50 ng/ml) gave a 3-fold increase of CD41a+CD42a+ MKs, with similar dose-dependent kinetics as TPO. IL-11 has also been reported to enhance MK development. To test whether FDA-approved pharmacological IL-11, Oprelvekin (Neumega®), further stimulated MK differentiation from hiPSCs, we cultured hematopoietic progenitor cells from day 14 in the presence of Romiplostium and Oprelvekin for 5 days. Our data showed that Romiplostium and Oprelvekin synergistically promote megakaryocytic differentiation. In the presence of Romiplostium, 60 to 95 % of cells were CD41a+CD42a+ MKs. Addition of Oprelvekin significantly increased the number of CD41a+CD42a+ MKs, but not the percentage of CD41a+CD42a+ MKs, suggesting that Oprelvekin enhanced a proliferation of MK progenitors. So far, 10 hiPSC lines from several individuals have been tested using the combination of Romiplostium and Oprelvekin in the feeder-free and serum-free differentiation condition. We are currently investigating if the MKs and platelets generated by this defined and scalable system are as fully functional as those generated from bone marrow CD34+ cells from healthy donors. * The first three authors contributed equally; This study is supported in part by an NIH grant U01 HL-107446 and 2012-MSCRFII-0124 (to ZZ Wang). Disclosures: No relevant conflicts of interest to declare.

Functional Early Hematopoietic Progenitor Cells Derived From Mouse Embryonic Stem Cells and Induced Pluripotent Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2421-2421 ◽  
Author(s):  
Cheng Li ◽  
Daniel R. George ◽  
Nichole M. Helton ◽  
Jeffery M. Klco ◽  
Jacqueline L. Mudd ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Progenitor Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Nsg Mice ◽  
Induced Pluripotent

Abstract We have previously reported a method to produce early hematopoietic progenitor cells from C57BL/6J-derived mouse embryonic stem cells (mESCs). After co-culture on OP9 stromal cells for one week, four different C57Bl/6 mESC lines consistently differentiated into hematopoietic progenitors, as determined by immunophenotyping; we detected cells that mark as KLS (Lin- Kit+ Sca+), CMPs, GMPs, and MEPs (but not SLAMs) from all four lines. In addition, functional progenitors for erythrocytes, monocytes, and mast cells (by morphology and immunophenotyping) were detected after another week of culture in methylcellulose with hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). These findings were replicated using four different lots of fetal bovine serum, and with three different lots of OP9 cells from ATCC. We injected 1x106 “OP9-induced” progenitor cells retroorbitally into unconditioned NSG mice, and detected multilineage hematopoietic engraftment (myeloid compartments marked by CD34, CD11b, Kit, and Gr-1, lymphoid compartments marked by CD3 and B220, and erythroid compartments marked by Ter119) in the bone marrow and/or spleens of 10 out of 19 recipients at 3 months. Using the OP9 co-culture system, we have differentiated miPSC clones from three independent iPSC experiments, using an integrating polycistronic lentivirus expressing OCT4, SOX2, and KLF4 as the reprogramming vector. One set of miPSC clones was produced from mouse embryonic fibroblasts (MEFs) from pooled C57BL/6J embryos, and two sets were made from adult mouse fibroblasts derived from a single animal, producing 6, 12, and 12 independent iPS clones for analysis, respectively. All thirty clones had pluripotent features, as determined by alkaline phosphatase staining and immunophenotyping (SSEA1, Oct4, and Nanog). We have injected the OP9-induced progenitors derived from one miPSC clone into NSG mice; thus far, 2 out of 14 recipients have demonstrated engraftment in the peripheral blood. However, the efficiency of hematopoietic progenitor generation with OP9 induction (based on the immunophenotyping and progenitor assays noted above) was highly variable for miPSCs from all three experiments. Among all three sets of miPSC clones, 18/30 exhibited differentiation efficiencies comparable to wild-type B6 ESCs, 5/30 clones exhibited moderately reduced differentiation efficiencies, 5/30 clones exhibited markedly reduced differentiation efficiencies, and 2/30 clones (from two different iPSC experiments) did not produce any detectable hematopoietic progenitors with OP9 induction. These phenotypes were stable and highly reproducible. The 2 clones that did not yield any hematopoietic progenitors had robust pluripotency marks, and one that was injected into the hindflank of NSG mice produced cystic teratomas. We found that 2% DMSO pretreatment of mESCs for 24 hours prior to OP9 co-culture improved the differentiation efficiency of wild-type B6 ESCs by 50% (Chetty et al. Nature Methods 10(6):553-6, 2013), but it did not rescue the phenotype of miPSC clones that did not produce hematopoietic progenitors. We are currently performing exome sequencing on the 24 miPSC clones from the adult fibroblast reprogramming experiments to determine whether phenotypic heterogeneity is due to specific mutations in the iPSC clones (Young et al. Cell Stem Cell 10(5):570-82, 2012). In summary, we have developed a simple system to derive functional early hematopoietic progenitor cells from mouse embryonic stem cells and/or induced pluripotent stem cells. OP9-induced progenitor cells engraft into NSG mice without the need for forced expression of HoxB4 (Wang et al. Proc Natl Acad Sci USA 102(52):19081-6, 2005). We have detected functional heterogeneity in miPSC clones derived from the same parental cells, which could be due to genetic variation in the founding cell from which each clone was derived, to different integration sites of the OSK lentivirus in each clone, or to as yet undefined epigenetic mechanisms. Exome sequencing may help to resolve this issue. Regardless, this approach could be a valuable tool for studying the hematopoietic development of a variety of mESC lines and/or miPSC lines derived from genetically altered mice. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Enhanced Hematopoiesis by Hematopoietic Progenitor Cells Lacking Intracellular Adaptor Protein, Lnk

2002 ◽  
Vol 195 (2) ◽  
pp. 151-160 ◽  
Author(s):  
Satoshi Takaki ◽  
Hatsue Morita ◽  
Yoshinari Tezuka ◽  
Kiyoshi Takatsu
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Adult Life ◽  
Adaptor Protein ◽  
Mitogen Activated Protein Kinase ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Mitogen Activated Protein

Hematopoietic stem cells (HSCs) give rise to variety of hematopoietic cells via pluripotential progenitors and lineage-committed progenitors and are responsible for blood production throughout adult life. Amplification of HSCs or progenitors represents a potentially powerful approach to the treatment of various blood disorders and to applying gene therapy by bone marrow transplantation. Lnk is an adaptor protein regulating the production of B cells. Here we show that Lnk is also expressed in hematopoietic progenitors in bone marrow, and that in the absence of Lnk, the number and the hematopoietic ability of progenitors are significantly increased. Augmented growth signals through c-Kit partly contributed to the enhanced hematopoiesis by lnk−/− cells. Lnk was phosphorylated by and associated with c-Kit, and selectively inhibited c-Kit–mediated proliferation by attenuating phosphorylation of Gab2 and activation of mitogen-activated protein kinase cascade. These observations indicate that Lnk plays critical roles in the expansion and function of early hematopoietic progenitors, and provide useful clues for the amplification of hematopoietic progenitor cells.

Stromal Microenvironment Regulates Progenitor Survival through Nitric Oxide Production.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1440-1440 ◽  
Author(s):  
Dima Jouni ◽  
Yanyan Zhang ◽  
Hakim Bouamar ◽  
Monika Wittner ◽  
William Vainchenker ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Culture Medium ◽  
Hematopoietic Cells ◽  
Hematopoietic Progenitor Cells ◽  
No Production ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor

Abstract Abstract 1440 Poster Board I-463 Nitric oxide (NO) is a small gaseous molecule with diverse roles including the regulation of cell proliferation, differentiation, apoptosis, adhesion and migration. NO is derived from L-arginine by the nitric oxide synthase (NOS) family of enzymes. At least three distinct NOS isoforms have been identified in mammalian cells including the endothelial (eNOS), neuronal (nNOS) and inducible (iNOS). Recently, we have shown that NO donors induce CXCR4 expression in human CD34 positive cells suggesting that NO production may regulate the migration and adhesion of hematopoietic progenitors. To determine the in vivo relevance of these findings and define the role of NO in the biology of hematopoietic progenitor cells, we first investigated NOS expression in hematopoietic and non-hematopoietic cells. Using quantitative reverse transcription-PCR analysis, we demonstrate that nNOS, and eNOS isoforms are highly expressed in Human Umbilical Vein Endothelial Cells (HUVEC) and osteoblastic cell lines, but there was no or low expression of NOS in hematopoietic cells including cell lines and mature blood cells except macrophages that express high level of iNOS. In agreement with RNA analysis, we found large amounts of nitrite (a stable derivative of NO) in the culture medium of stromal (MS-5) and osteoblasic (MG-63) cell lines. To determine the effects of NO on hematopoietic progenitor cells survival, cord blood CD34+ were cultured on MS-5 cells in the presence/absence of NOS inhibitor (L-NAME) for three days, then hematopoietic progenitor numbers were determined by culture in a semi-solid medium and colonies were quantified 14 days later. Treatment with L-NAME reduced colony number by 33, 42 and 54% with 10, 100 and 500 μM L-NAME concentrations, respectively. This effect is NO specific since the diminution of progenitor number was reverted by adding NO donor in the culture medium. These results indicate that NO is produced by bone marrow stromal cells. This production regulates the survival of hematopoietic progenitors in vitro. Further experiments are needed to determine if these effects are dependent on the regulation of CXCR4/SDF-1 signaling. Disclosures Jouni: Région IDF: Employment. Bouamar: Association pour la Recherche sur le Cancer: Employment; Cancéropôle IDF: Employment.

scholarly journals Hematopoietic defects in mice lacking the sialomucin CD34

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 479-490 ◽  
Author(s):  
J Cheng ◽  
S Baumhueter ◽  
G Cacalano ◽  
K Carver-Moore ◽  
H Thibodeaux ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Surface ◽  
Progenitor Cells ◽  
Es Cells ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Cell Surface Glycoprotein ◽  
Peripheral Cell

Although the pluripotent hematopoietic stem cell can only be definitively identified by its ability to reconstitute the various mature blood lineages, a diversity of cell surface antigens have also been specifically recognized on this subset of hematopoietic progenitors. One such stem cell-associated antigen is the sialomucin CD34, a highly O-glycosylated cell surface glycoprotein that has also been shown to be expressed on all vascular endothelial cells throughout murine embryogenesis as well as in the adult. The functional significance of CD34 expression on hematopoietic progenitor cells and developing blood vessels is unknown. To analyze the involvement of CD34 in hematopoiesis, we have produced both embryonic stem (ES) cells and mice that are null for the expression of this mucin. Analysis of yolk saclike hematopoietic development in embryoid bodies derived from CD34- null ES cells showed a significant delay in both erythroid and myeloid differentiation that could be reversed by transfection of the mutant ES cells with CD34 constructs expressing either a complete or truncated cytoplasmic domain. Measurements of colony-forming activity of hematopoietic progenitor cells derived from yolk sacs or fetal livers isolated from CD34-null embryos also showed a decreased number of these precursor cells. In spite of these diminished embryonic hematopoietic progenitor numbers, the CD34-null mice developed normally, and the hematopoietic profile of adult blood appeared typical. However, the colony-forming activity of hematopoietic progenitors derived from both bone marrow and spleen is significantly reduced in adult CD34-deficient animals, and these CD34-deficient progenitors also appear to be unable to expand in liquid cultures in response to hematopoietic growth factors. Even with these apparent progenitor cell deficiencies, CD34- null animals showed kinetics of erythroid, myeloid, and platelet recovery after sublethal irradiation that are indistinguishable from wild-type mice. These data strongly suggest that CD34 plays an important role in the formation of progenitor cells during both embryonic and adult hematopoiesis. However, the hematopoietic sites of adult CD34-deficient mice may still have a significant reservoir of progenitor cells that allows for normal recovery after nonmyeloablative peripheral cell depletion.

Reprogrammed Murine Fibroblasts Differentiated into Hematopoietic Progenitors Are Able to Successfully Engraft and Repopulate the Bone Marrow

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 389-389 ◽  
Author(s):  
Zaida Alipio ◽  
Dan Xu ◽  
Jianchang Yang ◽  
Louis M. Fink ◽  
Wilson Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitors

Abstract Cellular therapy using embryonic stem cells has always been an area of great interest due to the pluripotent characteristics of stem cells. In 2006, Takahashi and Yamanaka (Cell 126, 663–676) demonstrated that somatic cells can be reprogrammed into a stem cell-like state, termed induced pluripotent stem (iPS) cells, by ectopic expression of Oct4, Sox2, Klf4 and c Myc. A later report (Nakagawa et al. Nat. Biotechnol.26:101–106, 2008) showed that iPS cells can be produced in the absence of the c Myc oncogene. We have used this latter strategy to successfully reprogram somatic cells derived from C57BL/6 mouse tail fibroblast to iPS cells. Retrovirus infected fibroblasts exhibited stem cell-like morphology by 14 days post infection. These iPS cells were then infected with a retrovirus that expressed HOXB4. Recombinant leukemia inhibitor factor (LIF) supplement was removed from media at this time and the cells allowed to differentiate into embryoid bodies. These cells were screened for specific differentiation stem cell markers, such as Oct4, Nanog, Sall4 and SSEA-1. iPS cells were converted into embryonic bodies and then infected with retroviruses expressing HOXB4. Embryoid bodies stably expressing HOXB4 were induced to hematopoietic differentiation by treatment of thrombopoietin (TPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), interferon gamma (IFNg) and fms-like tyrosine kinase (FLT3 ligand). Evaluation of iPS-derived hematopoietic cells on smears show strikingly similarity in morphology to the W4 mouse embryonic stem (ES) cells differentiated into hematopoietic cells as a control. Flow cytometry analysis of iPS-derived hematopoietic cells after 1 week exposure to cytokines revealed 7% B220+ cells (B cells), 11% Ter119+ cells (erythroid), and 13% Gr-1+ cells (granulocytes) similar to W4 ES cells. The iPS-derived hematopoietic cells were transplanted into irradiated immunodeficient mice via lateral tail vein injection. Transplantation of these iPS-derived hematopoietic progenitors tagged with GFP into irradiated SCID mice revealed that the hematopoietic progenitors were able to home to the bone marrow after 1 week of transplantation. Importantly, after 1 month, GFP+ engrafted cells remained in the bone marrow suggesting a long-term engraftment. This long term engraftment of the iPS-derived hematopoietic cells to the bone marrow constitutes an important step toward potential therapy of numerous patient-specific blood based diseases.

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