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PubMed2004

Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells.

The first cell differentiation event in mammalian embryogenesis segregates inner cell mass lineage from the trophectoderm at the blastocyst stage. Oct-4, a member of the POU family of transcription factors, is necessary for the pluripotency of the inner cell mass lineage. Embryonic stem (ES) cells, which contribute to all of embryonic lineages, express the Oct-4 gene. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage in vitro, never contribute to embryonic proper tissues in chimeras and differentiate only into trophoblastic cells in the placenta. Expression of the Oct-4 gene was undetectable and severely repressed in trophoblastic lineage, including the stem cells. We found that the culture of TS cells with 5-aza-2'-deoxycytidine or trichostatin A caused the activation of the Oct-4 gene. Analysis of the DNA methylation status of mouse Oct-4 gene upstream region revealed that Oct-4 enhancer/promoter region was hypomethylated in ES cells but hypermethylated in TS cells. Furthermore, in vitro methylation suppressed Oct-4 enhancer/promoter activity in reporter assay. In the placenta of Dnmt1(n/n) mutant mice, most of the CpGs in the enhancer/promoter region were unmethylated, and Oct-4 gene expression was aberrantly detected. Chromatin immunoprecipitation assay revealed that Oct-4 enhancer/promoter region was hyperacetylated in ES cells compared with TS cells, thus demonstrating that DNA methylation status is closely linked to the chromatin structure of the Oct-4 gene. Here we propose that the epigenetic mechanism, consisting of DNA methylation and chromatin remodeling, underlies the developmental stage- and cell type-specific mechanism of Oct-4 gene expression.

Animals↗
PubMed2006

Reprogramming somatic cells into stem cells.

Recent scientific achievements in cell and developmental biology have provided unprecedented opportunities for advances in biomedical research. The demonstration that fully differentiated cells can reverse their gene expression profile to that of a pluripotent cell, and the successful derivation and culture of human embryonic stem cells (ESCs) have fuelled hopes for applications in regenerative medicine. These advances have been put to public scrutiny raising legal, moral and ethical issues which have resulted in different levels of acceptance. Ethical issues concerning the use of cloned human embryos for the derivation of stem cells have stimulated the search for alternative methods for reversing differentiated cells into multi/pluripotent cells. In this article, we will review the present state of these reprogramming technologies and discuss their relative success. We also overview reprogramming events after somatic cell nuclear transfer (SCNT), as they may further instruct ex ovo strategies for cellular manipulation.

Animals↗
PubMedDate not supplied

[Residual placental blood and bone marrow as sources of hematopoietic stem cells for allogenic stem cell transplantation. Comparative analysis of hematopoietic potential].

Umbilical cord blood (UCB) contains a high number of hematopoietic progenitor cells, which can be used for a HLA-identical transplantation. To proof the usefulness of this source of stem cells for transplantation we compared the cellular composition of 19 specimen of bone marrow of healthy donors (BM) and 23 of UCB. We determined the cell number, the frequency of CD34+ progenitor cells and the CFU-GM as well as the T- and B-cell-subsets by means of flow cytometry and in vitro culture methods. The T-cell content was higher in UCB than in BM while the B-cell number was lower. The CD34+ progenitor number per ml was 2.9 fold higher in BM than in UCB, but the frequency and the proliferative capacity of the CFU-GM progenitor cells in the CD34+ population was 8.6 fold higher in UCB than in BM. First clinical trials have demonstrated the usefulness of UCB for allogeneic stem cell transplantation in children. It is possible to harvest 80-100 ml UCB per placenta containing 1.2 x 10(9) nucleated cells with 9.7 x 10(5) (4-15 x 10(5)) CFU-GM.

Adolescent↗
PubMed2026

Effect of adding umbilical cord blood derived stem cells to haploidentical stem cell transplant (haplo-cord) on post-transplant survival and graft-versus-host disease in patients with hematological malignancies: A systematic review and meta-analysis.

BACKGROUND AND OBJECTIVES: Haploidentical stem cell transplantation (haplo-SCT) carries a substantial risk of graft-versus-host disease (GvHD), whereas umbilical cord blood (UCB) transplantation offers lower GvHD risk but slower engraftment. The haplo-cord approach combines both graft sources, aiming to mitigate GvHD while ensuring timely engraftment. This meta-analysis compares haplo-cord transplantation with haplo-SCT alone for the treatment of hematological malignancies. METHODS: Four electronic databases and two clinical trial registries were systematically searched. Effect sizes from eligible studies were pooled using odds ratios (ORs) for dichotomous outcomes and hazard ratios (HRs) for time-to-event outcomes. RESULTS: Twelve studies met the inclusion criteria. Haplo-cord was associated with a statistically significant reduction in chronic GvHD (OR = 0.62, 95%-CI: 0.42-0.93), while no significant difference was observed for grade II-IV acute GvHD (OR = 0.75, 95%-CI: 0.52-1.09). Survival outcomes favored haplo-cord, with lower HRs for overall survival (HR = 0.68, 95%-CI: 0.53-0.86) and event-free survival (HR = 0.61, 95%-CI: 0.52-0.72), while non-relapse mortality was not significant. Relapse at 3 years was significantly lower with haplo-cord (OR = 0.54, 95%-CI: 0.35-0.82). Haplo-cord also demonstrated higher day-30 engraftment, along with lower relapse-related and GvHD-related mortality, while CMV and EBV viremia showed no difference between groups. CD34 selection in the haplo graft significantly influenced effect sizes and heterogeneity in both subgroup analyses and meta-regression for acute GvHD. CONCLUSION: Haplo-cord transplantation improves GvHD outcomes, survival, and relapse risk compared with haplo-SCT alone. However, whether protocol optimization, possibly via CD34 selection, confers additional benefit remains uncertain and requires confirmation in future studies.

Humans↗
PubMed2005

Successful transplantation of haploidentically mismatched peripheral blood stem cells using CD133+-purified stem cells.

OBJECTIVE: For recipients of haploidentically mismatched stem cell allografts, T-cell depletion is mandatory to prevent lethal graft-vs-host disease (GVHD). Prevention of GVHD can be accomplished by negative selection of T cells or positive selection of stem cells. Recently, a new method for positive selection of stem cells was introduced using monoclonal antibodies against CD133 antigen. We report five cases of successful application of immunomagnetic separation of CD133+ stem cells for haploidentically mismatched allogeneic stem cell transplantation. METHODS: Five patients with high-risk hematological malignancies, ages 7 to 63 years old (median, 17 years), underwent peripheral blood stem cell transplantation from haploidentically mismatched related donors. Conditioning protocol was tailored according to patient clinical situation and included combination of treosulfan/fludarabine/thiotepa/melphalan/Mabcampath. Two patients did not get thiotepa. One of them received a protocol that included infusion of 4.4 x 10(7) blood mononuclear cells from the donor (day -9), followed by a combination of fludarabine/cyclophosphamide/busulfex/MabCampath. Separation of CD133+ stem cells was done using CliniMACS with Miltenyi's CD133 reagent. RESULTS: The procedure was well tolerated by all patients. Early 3-lineage engraftment was documented and none exhibited immune-mediated rejection. Time to recovery of absolute neutrophils count above 0.5 x 10(9)/L and 1.0 x 10(9)/L was 10 to 15 days (median, 14) and 11 to 29 days (median, 15), respectively. Time for platelet recovery to values greater than 20 x 10(9)/L and greater than 50 x 10(9)/L ranged from 12 to 25 days (median, 13.5), and from 14 to 34 days (median, 16), respectively. Transplant-related mortality did not occur in any of the patients. CONCLUSION: Our successful pilot trial suggests that positive selection of CD133+ stem cells may be a useful method for safe transplantation with haploidentically mismatched stem cell allografts while avoiding lethal acute and chronic GVHD. Future studies will be required to assess the clinical benefits of stem cell purification with CD133+ in comparison with CD34+ stem cells.

AC133 Antigen↗
PubMed2003

Label-retaining cells (presumptive stem cells) of mice vibrissae do not express gap junction protein connexin 43.

We investigated whether connexin 43, a gap junction protein present in human epidermis and mouse hair follicle, can serve as a negative marker for keratinocyte stem cells. Experiments carried out in mouse pelage and vibrissae hair follicles demonstrated that most of the slowly cycling cells, detected as label-retaining cells, do not express connexin 43. In humans, cells with immunohistochemically undetectable levels of connexin 43 are found in the epidermal basal layer of neonatal foreskin, and in the follicular bulge region. About 10% of the basal keratinocytes are connexin 43 negative, as determined by flow cytometry. These cells are uniformly small and low in granularity suggesting that presumptive keratinocyte stem cells can be identified and separated based on connexin 43 expression.

Animals↗
PubMed1986

Multi-type Galton-Watson process as a model for proliferating human tumour cell populations derived from stem cells: estimation of stem cell self-renewal probabilities in human ovarian carcinomas.

A mathematical model for proliferation of tumour cell populations is developed. The cell population is assumed to be organized in a hierarchy of decreasing proliferative potential and increasing degree of differentiation. Using some elements of the theory of Multi-type Galton-Watson processes, a method is proposed for the estimation of Psr, the probability of self-renewal of tumour stem cells, from the experimental distribution of clonal unit sizes obtained in cell culture studies. Six data sets from patients with advanced adenocarcinoma of the ovary are used to demonstrate the method. Reasonable estimates are obtained, and the theoretical colony size distributions predicted by the model appear to be in good qualitative agreement with the experimental ones, and lend support to a stem cell model of tumour growth. The possible significance of Psr as a prognostic factor is briefly discussed.

Cell Division↗
PubMed1976

Tumor virus effects on immunocyte precursor cells. Hemopoietic stem cell behavior and leukemogenic susceptibility.

Studies of Rauscher virus-induced erythroleukemia have demonstrated immunodepressive effects in the host and enhanced leukemogenesis with adjuvant administration. These observations led to the study of leukemic development in the NZB strain as a natural model of the experimentally adjuvant-stimulated animal. The results of such investigation would attribute the increased susceptibility of NZB mice to the possession of an enlarged population of pluripotent hemopoietic stem cells in active cell cycle. Studies with radiation chimeras have further shown that elevated endogenous spleen colony formation, the increased potential for autoimmunity, and for susceptibility to Rauscher viral leukemogenesis are all linked through the NZB hemopoietic system. It is concluded that the presence of an enlarged compartment of cyclically active stem cells may be an etiologic factor in the susceptibility to both virus-induced leukemia and the development of autoimmune disease.

Adjuvants, Immunologic↗
PubMed2002

Overexpression of the myeloid leukemia-associated Hoxa9 gene in bone marrow cells induces stem cell expansion.

Cytogenetic, genetic, and functional studies have demonstrated a direct link between deregulated Hoxa9 expression and acute myeloid leukemia (AML). Hoxa9 overexpression in mouse bone marrow cells invariably leads to AML within 3 to 10 months, suggesting the requirement for additional genetic events prior to AML. To gain further insight into how Hoxa9 affects hematopoietic development at the preleukemic stage, we have engineered its overexpression (1) in hematopoietic stem cells using retrovirus-mediated gene transfer and generated bone marrow transplantation chimeras and (2) in lymphoid cells using transgenic mice. Compared with controls, recipients of Hoxa9-transduced cells had an about 15-fold increase in transplantable lymphomyeloid long-term repopulating cells, indicating the capacity for this oncogene to confer a growth advantage to hematopoietic stem cells. In addition, overexpression of Hoxa9 in more mature cells enhanced granulopoiesis and partially blocked B lymphopoiesis at the pre-B-cell stage but had no detectable effect on T lymphoid development. Interestingly, despite specifically directing high expression of Hoxa9 in T and B lymphoid lineages, none of the Hoxa9 transgenic mice developed lymphoid malignancies for the observation period of more than 18 months.

Animals↗
PubMed2006

How signaling promotes stem cell survival: trophoblast stem cells and Shp2.

Trophoblast stem (TS) cells require FGF4 for self-renewal and to prevent differentiation. In this issue of Developmental Cell, Yang and colleagues show that the tyrosine phosphatase Shp2 prevents apoptosis in TS cells, by activation of Erk and subsequent phosphorylation and destabilization of the pro-apoptotic protein Bim. These studies provide a novel link between FGF/Erk signaling and cell survival that may be relevant to other stem and progenitor cell niches.

Animals↗
PubMed1995

Long-term culture of chronic myelogenous leukemia marrow cells on stem cell factor-deficient stroma favors benign progenitors.

Long-term culture of marrow from patients with chronic myelogenous leukemia (CML) has been reported to favor the outgrowth of bcr/abl- progenitor cells in some patients. We examined the effect of the presence of soluble or transmembrane forms of stem cell factor (SCF) in long-term cultures of CML marrow. CD34-enriched cells from CML patients in advanced chronic phase or accelerated phase were plated on immortalized fetal liver stromal cells from homozygous SCF-deficient SI/SI mice (SI/SI4) with or without the addition of soluble human SCF, SI/SI4 cells expressing high levels of the transmembrane form of human SCF (SI/SIh220), or primary human allogeneic stroma. Cells were removed from cultures and plated weekly in colony assays. The clonagenic cell output from cultures completely lacking SCF was lower over the first 2 to 3 weeks, but by 5 weeks was similar to the clonagenic cell output from the other culture conditions. Analysis of bcr/abl transcripts from individual colonies showed a lower percentage of malignant progenitors present in long-term cultures completely deficient in SCF than under the other culture conditions, particularly compared with primary human stroma-containing long-term cultures. SCF may specifically favor malignant versus benign progenitor cells present in the marrow of CML patients, and an abnormal proliferative response to SCF in very primitive cells may be an underlying defect in the pathophysiology of this disease.

Adult↗
PubMed2006

Capillary morphogenesis gene (CMG)-1 is among the genes differentially expressed in mouse male germ line stem cells and embryonic stem cells.

We recently established a technique to expand male germ line stem (GS) cells in long-term culture without losing their spermatogenic capacity. To gain insight into the genetic program of these cells, we compared the mRNA expression profile of GS cells with that of embryonic stem (ES) cells using DNA microarrays. We found 79 genes that were upregulated in GS cells compared to ES cells, including synaptonemal complex protein-1, deleted in azoospermia-like, ubiquitin-conjugating enzyme E2B, and ubiquitin carboxy-terminal hydrolase L1, all of which are functionally important for spermatogenesis. In addition, we identified a cDNA encoding the mouse ortholog of capillary morphogenesis gene (CMG)-1. CMG-1 transcripts were predominantly produced in spermatogonia and spermatocytes in mouse testis. When CMG-1 expression was attenuated in a mouse spermatocyte-derived cell line, GC-2spd(ts), by a target-specific short interfering RNA, the morphology of the cells was changed and the expression of cyclin D2 was abrogated. A reporter assay using a genomic region upstream of the mouse cyclin D2 gene revealed that this downmodulation occurs at the transcriptional level. We detected FLAG-tagged CMG-1 protein in the nuclei of transfected COS7 cells, suggesting that CMG-1 may play a unique role in the transcriptional regulation of the cyclin D2 gene. The upregulated GS genes identified in this study will provide useful information for the future investigation of spermatogonial stem cells and the early phase of male germ cell differentiation.

Animals↗
PubMed2006

Fyn kinase acts upstream of Shp2 and p38 mitogen-activated protein kinase to promote chemotaxis of mast cells towards stem cell factor.

The c-Kit receptor protein-tyrosine kinase plays a critical role in the differentiation, growth and survival of mast cells. Binding of its ligand stem cell factor (SCF), induces c-Kit dimerization, autophosphorylation, and recruitment of signaling proteins. The juxtamembrane sequence of c-Kit contains recruitment sites for the Src family kinases Fyn and Lyn, as well as Shp1 and Shp2 protein-tyrosine phosphatases. To characterize the role of Fyn in c-Kit signaling, we generated bone marrow-derived mast cells (BMMCs) from wild-type and Fyn knock-out mice. In contrast with previous studies of Lyn-deficient BMMCs, SCF treatment of Fyn-deficient BMMCs revealed no overt defects in the overall pattern of tyrosine phosphorylation, phosphatidylinositol 3' kinase recruitment to c-Kit, or phosphorylation of Stat3 transcription factor. However, Fyn-deficient mast cells showed a significant reduction in phosphorylation of Shp2 phosphatase and p38 mitogen-activated protein kinase. Defects in Shp2 and p38 phosphorylation were restored in Fyn-deficient mast cells transduced with a Fyn-expressing retrovirus (Fyn-rescue). Fyn-deficient BMMCs displayed reduced chemotaxis towards SCF, and this defect was corrected in Fyn-rescue cells. This study provides evidence that recruitment of both Shp2 and Fyn to juxtamembrane sites in c-Kit results in Shp2 phosphorylation, downstream signaling to p38 mitogen-activated protein kinase, and enhanced chemotaxis of mast cells.

Animals↗
PubMed2005

The Drosophila ovarian and testis stem cell niches: similar somatic stem cells and signals.

The stem cell niches at the apex of Drosophila ovaries and testes have been viewed as distinct in two major respects. While both contain germline stem cells, the testis niche also contains "cyst progenitor" stem cells, which divide to produce somatic cells that encase developing germ cells. Moreover, while both niches utilize BMP signaling, the testis niche requires a key JAK/STAT signal. We now show, by lineage marking, that the ovarian niche also contains a second type of stem cell. These "escort stem cells" morphologically resemble testis cyst progenitor cells and their daughters encase developing cysts before undergoing apoptosis at the time of follicle formation. In addition, we show that JAK/STAT signaling also plays a critical role in ovarian niche function, and acts within escort cells. These observations reveal striking similarities in the stem cell niches of male and female gonads, and suggest that they are largely governed by common mechanisms.

Animals↗
PubMed2006

Differentiation of human embryonic stem cells into bipotent mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors that can be found in many connective tissues, including fat, bone, cartilage, and muscle. We report here a method to reproducibly differentiate human embryonic stem cells (hESCs) into MSCs that does not require the use of any feeder layer. The cells obtained with this procedure are morphologically similar to bone marrow MSCs, are contact-inhibited, can be grown in culture for about 20 to 25 passages, have an immunophenotype similar to bone marrow MSCs (negative for CD34 and CD45 and positive for CD13, CD44, CD71, CD73, CD105, CD166, human leukocyte antigen [HLA]-ABC, and stage-specific embryonic antigen [SSEA]-4), can differentiate into osteocytes and adipocytes, and can be used as feeder cells to support the growth of undifferentiated hESCs. The ability to produce MSCs from hESCs should prove useful to produce large amounts of genetically identical and genetically modifiable MSCs that can be used to study the biology of MSCs and for therapeutic applications.

Cell Differentiation↗
PubMed2000

Effective ex vivo generation of megakaryocytic cells from mobilized peripheral blood CD34(+) cells with stem cell factor and promegapoietin.

OBJECTIVE: The additional transplantation of ex vivo-generated megakaryocytic cells might enable the clinician to ameliorate or abrogate high-dose chemotherapy-induced thrombocytopenia. Therefore, the ex vivo expansion of CD34(+) PBPC was systematically studied aiming for an optimum production of megakaryocytic cells. MATERIALS AND METHODS: CD34(+) PBPC were cultured in serum-free medium comparing different (n = 23) combinations of stem cell factor (SCF) (S), IL-1beta (1), IL-3 (3), IL-6 (6), erythropoietin (EPO) (E), thrombopoietin (TPO) (T) and promegapoietin (PMP, a novel chimeric IL-3/TPO receptor agonist). Ex vivo-generated cells were assessed by flow cytometry, morphology, and progenitor cell assays. RESULTS: Addition of TPO to cultures stimulated with S163E, a potent progenitor cell expansion cocktail previously described by our group, effectively induced the generation of CD61(+) cells (day 12: 31.4 +/- 7.9%). The addition of PMP tended to be more effective than TPO +/- IL-3. Whereas EPO was not required to maximize TPO- or PMP-induced megakaryocytic cell production, the use of IL-6 and IL-1beta augmented cellular expansion as well as CD61(+) cell production rates in the majority of cytokine combinations studied. Thus, the most effective CD61(+) cell expansion cocktail consisted of S163 + PMP which resulted in 65.9 +/- 3.0% CD61(+) at day 12 and an overall production of 40.7 +/- 4.5 CD61(+) cells per seeded CD34(+) PBPC. However, the basic 2-factor combination S + PMP also allowed for an effective CD61(+) cell production (day 12 CD61(+) cell production: 15.1 +/- 1.6). Moreover, maximum amplification of CFU-Meg was observed after 7 days using this two-factor cocktail (12.9 +/- 2.6-fold). The majority of CD61(+) cells generated in TPO- or PMP-based medium were low-ploidy 4N and 8N cells, and ex vivo-generated CD61(+), CD41(+), and CD42b(+) cells were mainly double positive for FACS-measured intracellular von Willebrand Factor (vWF) (76.7 +/- 3.3%, 58.8 +/- 4.4%, and 82.7 +/- 2.5%, respectively). CONCLUSIONS: Taken together, this study demonstrates that megakaryocytic cells can be effectively produced ex vivo with as little as two-factors (SCF + PMP), an approach that might be favorably employed in a clinical expansion trial aiming to ameliorate high-dose chemotherapy-induced thrombocytopenia.

Cell Differentiation↗
PubMed2002

BCR-ABL alters the proliferation and differentiation response of multipotent hematopoietic cells to stem cell factor.

Chronic myeloid leukaemia (CML), a hematopoietic stem cell disorder is characterized by the expression of BCR-ABL. To investigate the effects of BCR-ABL on multipotent hematopoietic cells, a temperature sensitive BCR-ABL tyrosine kinase was expressed in the cell line, FDCP-Mix. BCR-ABL mediated an increase in c-kit expression that correlated with an enhanced mitogenic response to SCF. This was not observed in the absence of Bcr-Abl kinase activity or presence of the BCR-ABL inhibitor STI571, which also inhibits c-kit. When cultured in a combination of SCF plus G-CSF the FDCP-Mix cells undergo neutrophilic differentiation over a 7-10 day period. When BCR-ABL was active there was a marked inhibition of cell maturation compared to control cells in which BCR-ABL was either inactive or not present. However, BCR-ABL did not block differentiation as the cells eventually undergo terminal maturation. These data argue that BCR-ABL is directly responsible for the enhanced response to SCF reported in CML progenitor cells. Furthermore, although the primary effect of STI571 is via direct inhibition of BCR-ABL, STI571 additionally reduces the enhanced response to SCF. Thus there are two sites of STI571 action of potential importance in Bcr-Abl expressing cells.

Antineoplastic Agents↗