SDF-1/CXCL12 Enhances Survival of Murine Embryonic Stem Cells and Their Production of Myeloid Progenitors, but Not Hemangioblasts.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1693-1693
Author(s):  
Ying Guo ◽  
Giao Hangoc ◽  
Huimin Bian ◽  
Louis M. Pelus ◽  
Hal E. Broxmeyer
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Receptor Expression ◽  
Hematopoietic Progenitor Cells ◽  
Es Cell ◽  
Hematopoietic Progenitor ◽  
Myeloid Progenitors

Abstract Stromal cell derived factor (SDF)-1/CXCL12 has been shown to promote the survival of embryonic retinal ganglion cells, peritoneal B1a (PerBla) lymphocytes and chronic lymphocytic leukemia B cells. Our previous studies implicated SDF-1 as an important factor in enhancing survival of murine bone marrow (BM) hematopoietic stem cells and, human cord blood and adult human BM myeloid progenitors. Since pluripotent embryonic stem (ES) cells can give rise to differentiated cell types derived from all three primary germ layers (endoderm, mesoderm, and ectoderm) and adult stem cells are generated during embryoid body (EB) formation, we investigated whether SDF-1 has effects on survival of ES cells and EB generation of hematopoietic progenitor cells. In order to establish SDF-1 expression patterns during EB formation, we screened supernatants during day 1–5 EB formation for SDF-1 production by three murine ES cell lines (E14, R1 and CCE). We observed low but detectable SDF-1 secreted in cultures of ES cells and day 1 stage EBs. SDF-1 was increased in the media from day 2 stage EBs and continued to increase through days 4–5. CXCR4, SDF-1 receptor, expression was also analyzed. CXCR4 mRNA expression was low in ES cells and day 1–3 EBs, and increased significantly from Day 4 EBs, reaching maximum levels at day 5, and decreasing after day 6. Surface CXCR4 expression was consistent with mRNA data. To determine if SDF-1 had an effect on ES cell survival, we cultured ES cells without serum, and added serum at either 0, 24, 48 or 96 hrs to each of the following groups: A) Control, B) SDF-1 (100ng/ml) or C) AMD3100 (1 μM), an SDF-1 receptor (CXCR4) antagonist. Colonies were scored 7 days after the addition of serum. SDF-1 enhanced survival of ES cells, while AMD3100 decreased survival. We also checked the apoptosis of ES cells after withdrawing serum for 24, 48, 72 and 96 hours in four groups: A) control, B) SDF-1 (100ng/ml), C) AMD3100 (1 μM) or D) AMD3100 (1 μM) and SDF-1 (100ng/ml). SDF-1 decreased apoptosis and AMD3100 blocked the SDF-1 effect. AMD3100 alone increased apoptosis compared to control. This suggests that AMD3100 blocked endogenous SDF-1 effects. To determine if SDF-1 had an effect on differentiation of hematopoietic progenitor cells, we added SDF-1 (100 ng/ml), AMD3100, or AMD3100 plus SDF-1 (100 ng/ml) at the beginning of EB formation, immediately after removal of LIF, and quantitated primitive erythroid (p-BFU-E), definitive erythroid (d-BFU-E), granulocyte-macrophage (CFU-GM) and multipotential Granulocyte/Erythroid/Macrophage/Megakaryocyte (CFU-GEMM) colony formation. In comparison to control cells (cultured without SDF-1 and AMD3100), SDF-1 increased numbers of p-BFU-E, d-BFU-E, CFU-GM, and CFU-GEMM colonies. Addition of AMD3100 with SDF-1 blocked the enhancing effect of SDF-1. In addition, significantly decreased numbers of colonies were also observed in the presence of AMD3100 alone. This suggests that AMD3100 blocks endogenous SDF-1 actions, consistent with our data on SDF-1 production during EB formation. In order to determine when SDF-1 starts affecting hematopoiesis, hemangioblast colony assays were used. Neither SDF-1 nor AMD3100 influenced hemangioblast colony formation or expression of Flk-1 mRNA, a marker of hemangioblasts. The results suggest a role for SDF-1 in ES cell growth and differentiation.

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 Hematopoiesis and immunity of HOXB4-transduced embryonic stem cell–derived hematopoietic progenitor cells

Blood ◽  
2008 ◽  
Vol 111 (6) ◽  
pp. 2953-2961 ◽  
Author(s):  
Kun-Ming Chan ◽  
Sabrina Bonde ◽  
Hannes Klump ◽  
Nicholas Zavazava
Keyword(s):  
T Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Interleukin 2 ◽  
Embryonic Stem ◽  
Hematopoietic Progenitor Cells ◽  
Mixed Chimerism ◽  
Specific Response ◽  
Hematopoietic Progenitor ◽  
Immunodeficient Mice

Abstract The ability of embryonic stem (ES) cells to form cells and tissues from all 3 germ layers can be exploited to generate cells that can be used to treat diseases. In particular, successful generation of hematopoietic cells from ES cells could provide safer and less immunogenic cells than bone marrow cells, which require severe host preconditioning when transplanted across major histocompatibility complex barriers. Here, we exploited the self-renewal properties of ectopically expressed HOXB4, a homeobox transcription factor, to generate hematopoietic progenitor cells (HPCs) that successfully induce high-level mixed chimerism and long-term engraftment in recipient mice. The HPCs partially restored splenic architecture in Rag2−/−γc−/−–immunodeficient mice. In addition, HPC-derived newly generated T cells were able to mount a peptide-specific response to lymphocytic choriomeningitis virus and specifically secreted interleukin-2 and interferon-γ upon CD3 stimulation. In addition, HPC-derived antigen presenting cells in chimeric mice efficiently presented viral antigen to wild-type T cells. These results demonstrate for the first time that leukocytes derived from ES cells ectopically expressing HOXB4 are immunologically functional, opening up new opportunities for the use of ES cell–derived HPCs in the treatment of hematologic and immunologic diseases.

Hematopoietic Progenitor Cells Derived from Embryonic Stem Cells: Analysis of Gene Expression

Stem Cells ◽  
2002 ◽  
Vol 20 (5) ◽  
pp. 428-437 ◽  
Author(s):  
Shi-Jiang Lu ◽  
Chengshi Quan ◽  
Fei Li ◽  
Loyda Vida ◽  
George R. Honig
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Progenitor Cells ◽  
Embryonic Stem ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor

miRNA 125b Enhances the differentiation and Functionality of in Vitro Generated Human Hematopoietic Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1217-1217
Author(s):  
Julian Pulecio ◽  
Emmanuel Nivet ◽  
Marianna Vitaloni ◽  
Ignacio Sancho Martinez ◽  
Leire Abalde ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Progenitor Cells ◽  
Human Fibroblasts ◽  
Embryonic Stem ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Micro Rnas

Abstract Abstract 1217 The discovery of transcription factors and micro RNAs (miRNAs) capable of converting somatic cells, either into cells from different lineages or embryonic stem-like cells, has opened a road to differentiate and produce in vitro engraftable cells that could replace and fix damaged tissues. One of the most attractive and promising fields is the differentiation towards blood, considering it is a tissue without a complex tridimensional structure and that the phenotypes of the different sublineages are already well characterized. We previously reported the differentiation from induced pluripotent stem cells (iPS) towards hematopoietic cells and their derived lineages in in vitro assays. More recently, overexpression of Oct4 has been shown to allow for the transdifferentiation of human fibroblasts (HF) into blood progenitor cells albeit with compromised lymphoid differentiation capacity. However, there are no reports of successful differentiation into blood progenitors which are able to completely recover functionally immunodepleted mice. Considering the emerging role of miRNAs controlling development and differentiation of diverse cell lineages, we hypothesized that the gap missing in the current protocols to obtain hematopoietic stem cells can be filled by the data obtained by miRNA profiling of hematopoietic progenitor cells found in human Cord Blood samples (CB-HPC), which could unveil the key players in the maintenance of blood stemness. After in vitro expansion of different CB-HPC subpopulations, we have found a set of miRNAS specifically upregulated in the CD34+/CD90+ fraction, a population that has shown the highest capacity to repopulate immunodepleted mice. Upon serial testing, we found that miR-125b, previously reported to have a key role in human and murine hematopoiesis, is highly upregulated in CD34+/CD90+ CB-HPC. In particular, differentiation of iPS towards blood progenitors demonstrated that miR-125b overexpression, specifically increases the percentage and number of blood progenitors, as well as the ability to produce hematopoietic colonies after colony forming unit assay (CFU-A). In addition, following the same rationale previously reported for direct conversion of HF into blood progenitors, we overexpressed one of the transcription factors described by Yamanaka's group. The blood progenitors we obtained were deficient for the differentiation towards CD45+ cells, production of colonies in CFU-A, and showed no repopulation of immunodepleted mice. Most interestingly, the overexpression of miR-125b during the HF conversion, allowed us to obtain blood progenitors able to circumvent these deficiencies. Our preliminary results indicate that miR-125b overexpression causes a positive effect in the maintenance of blood progenitors through the downregulation of p53, a previously reported target for this miRNA in hematopoiesis. While several aspects remain to be addressed in terms of the prospective therapeutical approaches, our study proves that overexpression of miR-125b strongly enhances the differentiation and functionality of in vitro generated HPC. Disclosures: No relevant conflicts of interest to declare.

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