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Results for “stem cell differentiation”

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PubMed1976

Pluripotential stem cell differentiation in hemopoietic colonies.

To determine if mononuclear cells proliferating in murine hemopoietic spleen colonies were pluripotential in addition to possessing kinetic features of stem cells, we performed sequential studies of mice during their recovery from a split-dose irradiation regimen of 850 roentgens leg shielded-3-hr interval-850 roentgens leg irradiated (850R L.S. 3- L.I.). Injecting tritiated thymidine during stem cell compartment repletion 3 and 4 days after 850R L.S. 3- L.I. resulted in heavily labeled mononuclear cells resembling medium to large leptochromatic lymphocytes in the portion of spleen removed an hour after injection. The splenic remnant obtained from the same mouse 24-48 hr later contained lightly labeled erythroblasts, myeloid cells, and lymphoid cells. Grain counts suggested that erythroblasts and their precursors had undergone about four divisions, myeloid cells and their precursors two to three divisions, and lymphoid cells and their precursors two to three divisions during the 48-hr period. Similar studies in plethoric mice demonstrated the labeling of mononuclear cells on day 4 and their differentiation to myeloid and lymphoid cells by day 6. This finding confirmed that the labeled mononuclear cells were not exclusively erythroblast progenitors. On the basis of these and previous studies of post-irradiation survival and erythropoietic recovery, we conclude that these endogenous monomuclear cells, which resemble medium to large leptochromatic lymphocytes and replicate during stem cell compartment repletion, are pluripotential hemopoietic stem cells.

Animals↗
PubMed1998

Natural killer and B-lymphoid potential in CD34+ cells derived from embryonic stem cells differentiated in the presence of vascular endothelial growth factor.

Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels, differentiation intermediates were investigated. ES cells generated progeny expressing CD34, which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7, the CD34+ cells developed two types of B220+ CD34- lymphocytes: CD3- cytotoxic lymphocytes and CD19+ pre-B cells, and such lymphoid potential was highly enriched in the CD34+ population. Interestingly, the cytotoxic cells expressed the natural killer (NK) cell markers, such as NKR-P1, perforin, and granzymes, classified into two types, one of which showed target specificity of NK cells. Thus, ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells, and both myeloid and lymphoid cells seem to be derived from the CD34+ intermediate, on which VEGF may play an important role.

Animals↗
PubMed2001

The regulation of embryonic stem cell differentiation by leukaemia inhibitory factor (LIF).

LIF (leukaemia inhibitory factor) is commonly used to maintain mouse embryonic stem cells in an undifferentiated state. These cells spontaneously differentiate when allowed to aggregate in the absence of LIF, forming embryoid bodies in which early embryonic cell lineages develop. Using embryoid bodies cultured in the presence and absence of LIF, we show that although LIF inhibited the development of visceral and parietal endodermal cells, it did not affect the differentiation of the primitive endodermal cell precursors of these extraembryonic cell lineages. Furthermore, deposition of the basement membrane produced by the primitive endodermal cells, which separates them from the remaining cells of the embryoid body, still occurred. The differentiation of primitive ectodermal cells and their progeny was inhibited by LIF, as evidenced by the lack of expression of FGF-5, muscle, and neuronal markers. However, cavitation of the embryoid body and maintenance of the cells in contact with the primitive endodermal basement membrane as an epiblast epithelium still occurred normally in the presence of LIF. These results indicate that cavitation and formation of the epiblast epithelium are regulated by mechanisms distinct from those controlling the differentiation of epiblast cell lineages. Furthermore, although epithelium formation and cavitation do not require the differentiation of visceral endodermal cells, the results are consistent with the hypothesis that the primitive endodermal basement membrane is sufficient to induce the epithelialization of undifferentiated embryonic stem cells necessary for cavitation.

Animals↗
PubMed2005

Wnt4-transformed mouse embryonic stem cells differentiate into renal tubular cells.

Embryonic stem (ES) cells have the potential to differentiate into various progenitor cells. Here we investigated the capacity of mouse ES cells to differentiate into renal tubular cells both in vitro and in vivo. After stably transfecting Wnt4 cDNA to mouse ES cells (Wnt4-ES cells), undifferentiated ES cells were incubated by the hanging drop culture method to induce differentiation to embryoid bodies (EBs). During culturing of the EBs derived from the Wnt4-ES cells, aquaporin-2 (AQP2) mRNA and protein were expressed within 15-20 days. The expression of AQP2 in Wnt4-EBs was enhanced in the presence of hepatocyte growth factor (HGF) and activin A. We next performed in vivo experiments by transplanting the Wnt4-EBs into the mouse renal cortex. Four weeks after transplantation, some portions of the EB-derived cells expressing AQP2 in the kidney assembled into tubular-like formations. In conclusion, our in vitro and in vivo experiments revealed two new findings: first, that cultured Wnt4-EBs have an ability to differentiate into renal tubular cells; and second, that Wnt4, HGF, and activin A may promote the differentiation of ES cells to renal tubular cells.

Activins↗
PubMed1985

Reconstitution of the W/Wv stem cell differentiation defect by infection with Rauscher leukemia virus.

Hematopoietic stem cells of W/Wv mice failed to produce macroscopically visible hematopoietic spleen colonies in irradiated recipient mice. Infection of W/Wv mice of the spleen focus-forming virus-susceptible genotype Fv-2ss (DBA/2) or Fv-2rs (BD2F1) with Rauscher leukemia virus (RLV) restored the spleen colony-forming capacity of the stem cells. The resulting spleen colonies had normal size and cellularity; the frequency of and ratio between granulocyte-macrophage and erythroid progenitor cells were also normal, without excessive production of erythroid cells. The frequency of spleen colony-forming units (CFU-S) appeared to be strongly reduced in W/Wv mice. The seeding fraction of RLV-infected W/Wv stem cells in the recipient spleens did not differ from that of uninfected or RLV-infected +/+ stem cells. At equivalent numbers of CFU-S, spleen suspensions of RLV-infected W/Wv mice were equally effective as +/+ control suspensions in protecting irradiated mice from death due to bone marrow failure. Thus the number of CFU-S observed appeared to be predictive for the number of W/Wv cells required for effective radioprotection. In irradiated W/Wv mice that received transplants of RLV-infected W/Wv cells, circulating erythrocyte numbers approached those of control mice; the erythrocytes were of normal size, in contrast to the macrocytic red cells of untreated W/Wv mice. The reduced frequency of CFU-S in RLV-infected W/Wv mice can be readily explained by a reduced self-replicating capacity, attributable to the W/Wv genes, which was not reconstituted by infection with RLV. The data indicate a direct involvement of pluripotent stem cells upon infection with RLV.

Anemia, Macrocytic↗
PubMed2026

Stem Cell Differentiation Disperses Transcriptional Clusters via a Conserved Surface-Condensate Trajectory.

Stem cells exhibit exceptionally prominent transcriptional clusters, which dissolve with progressing differentiation. Although these clusters are assigned central roles in embryonic gene regulation, their formation and loss during differentiation remain poorly understood. This study reveals that these prominent clusters disperse along a conserved trajectory in mouse embryonic stem cells, fruit fly testes, and zebrafish embryos. Imaging and lattice simulations show that these clusters form via surface condensation on H3K27ac-marked super-enhancer regions, which act as genomic scaffolds. Upon differentiation, partial loss of these active epigenetic marks and transcription-driven unfolding lead to dispersal of the prominent clusters. The block copolymer-based lattice simulations explain this process as a conserved trajectory through a three-dimensional state space, governed by surface condensation principles that extend beyond canonical liquid-liquid phase separation. This work marks surface condensation as a biophysical mechanism for the dynamic organization of stem cell-specific transcriptional hubs and demonstrates evolutionary conservation in several organisms. By uncovering a conserved biophysical mechanism for transcriptional organization in development, our work illustrates how polymer properties can contribute to the control of cell identity and fate.

Animals↗
PubMed1985

Trophectodermal carcinoma: mouse teratocarcinoma-derived tumour stem cells differentiating into trophoblastic and yolk sac elements.

The transplantable tumour line derived from a spontaneous ovarian murine teratocarcinoma (Fekete & Ferigno, 1952) was cloned and characterized using light and electron microscopic and immunohistochemical techniques. Grown in ascites, the tumour consisted predominantly of stem cells and a small number of differentiated derivatives. The stem cells expressed surface reactivity with antibody to SSEA-3 and Forssman antigen, alkaline phosphatase, focal cytoplasmic reactivity with antibody to SSEA-1, and varying amounts of cytoplasmic glycogen and 3 beta-hydroxysteroid dehydrogenase. Their cytoskeleton reacted with antibodies to keratin and vimentin. The differentiated derivatives formed approximately 5-15% of the total cell population in ascites and appeared either as giant cells or were characterized by their reactivity with antibodies to H-2 or alpha-foetoprotein or intracellular and pericellular laminin or high levels of 3 beta-hydroxysteroid dehydrogenase activity. Solid tumours produced from subcutaneously injected cells had a variegated appearance suggesting, that like the limited differentiation in the ascites, the stem cells can give rise to trophoblastic, as well as parietal and visceral yolk sac elements. On the basis of the presented data the tumour stem cells were considered as representing malignant equivalents of the common precursor of trophoblastic, visceral and parietal yolk sac cells most likely corresponding to trophectoderm. Accordingly, the tumour was designated as trophectodermal carcinoma.

Animals↗
PubMed2002

Induction of dental pulp stem cell differentiation into odontoblasts by electroporation-mediated gene delivery of growth/differentiation factor 11 (Gdf11).

The long-term goal of dental treatment is to preserve teeth and prolong their function. In dental caries an efficient method is to cap the exposed dental pulp and conserve the pulp tissue with reparative dentin. We examined whether growth/differentiation factor 11 (GDF11), a morphogen could enhance the healing potential of pulp tissue to induce differentiation of pulp stem cells into odontoblasts by electroporation-mediated gene delivery. Recombinant human GDF11 induced the expression of dentin sialoprotein (Dsp), a differentiation marker for odontoblasts, in mouse dental papilla mesenchyme in organ culture. The Gdf11 cDNA plasmid which was transferred into mesenchymal cells derived from mouse dental papilla by electroporation, induced the expression of Dsp. The in vivo transfer of Gdf11 by electroporation stimulated the reparative dentin formation during pulpal wound healing in canine teeth. These results provide the scientific basis and rationale for gene therapy for endodontic treatments in oral medicine and dentistry.

Animals↗
PubMed2004

APC dosage effects in tumorigenesis and stem cell differentiation.

It is well established that concentration gradients of signaling molecules (the so-called "morphogens") organize and pattern tissues in developing animals. In particular, studies in Drosophila and different vertebrates have shown that gradients of the Wnt, Hedgehog (Hh) and transforming growth factor-beta (TGF-beta) families of morphogens play critical roles in limb patterning. Morphogens are often expressed in organizing centres and can act over a long range to coordinate the patterning of an entire field of cells. These observations imply that exposure to different concentrations of these diffusible factors may trigger differential cellular responses. In order to study these dosage-dependent Wnt/beta-catenin signaling effects, we have generated several hypomorphic mutant alleles at the mouse Apc locus and studied their cellular and phenotypic outcomes in stem cell renewal and differentiation, and in tumorigenesis. The results clearly show that Apc mutations differentially affect the capacity of stem cells to differentiate in a dosage-dependent fashion. Likewise, different Apc mutations (and the corresponding Wnt signaling dosages) confer different degrees of susceptibility to tumorigenesis in the corresponding mouse models. These results have implications for the understanding of the molecular and cellular basis of tumor initiation by defects in the Wnt pathway. We propose a model in which adult somatic stem cell compartments are characterized by tissue-specific beta-catenin threshold levels for cell proliferation, differentiation and apoptosis. Different APC mutations will result in different levels of beta-catenin signaling, thus conferring different degrees of tumor susceptibility in different tissues. Hence, beta-catenin dosage-dependent effects may not only explain how a single pathway is involved in the development and homeostasis of different tissues, but also its pleiotrophic role in tumorigenesis.

Adenomatous Polyposis Coli Protein↗
PubMed2006

Three-dimensional culture for expansion and differentiation of mouse embryonic stem cells.

Differentiation of embryonic stem (ES) cells typically requires cell-cell aggregation in the form of embryoid bodies (EBs). This process is not very well controlled and final cell numbers can be limited by EB agglomeration and the inability to drive differentiation towards a desired cell type. This study compares three-dimensional (3D) fibrin culture to conventional two-dimensional (2D) suspension culture and to culture in a semisolid methylcellulose medium solution. Two types of fibrin culture were evaluated, including a PEGylated fibrin gel. PEGylation with a difunctional PEG derivative retarded fibrinogen migration during through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a result of crosslinking, similarly, degradation was slowed in the PEGylated gel. ES cell proliferation was higher in both the fibrin and PEGylated fibrin gels versus 2D and methylcellulose controls. FACS analysis and real-time-PCR revealed differences in patterns of differentiation for the various culture systems. Culture in PEGylated fibrin or methylcellulose culture demonstrated features characteristic of less extensive differentiation relative to fibrin and 2D culture as evidenced by the transcription factor Oct-4. Fibrin gels showed gene and protein expression similar to that in 2D culture. Both fibrin and 2D cultures demonstrated statistically greater cell numbers positive for the vascular mesoderm marker, VE-cadherin.

Animals↗
PubMed2006

[Plasticity of bone marrow mesenchymal stem cells differentiating into cardiomyocytes and the potential of cardiac therapeutics].

Current treatments for ischemic cardiomyopathy aim at minimizing the deteriorated consequences of damaged myocardium. Recent stem cell researches have provided major impetus for the possibility of treating heart failure by generating new myocardium and vascular structures. Bone marrow mesenchymal stem cells (MSCs) , also referred to as marrow stromal cells, differentiate readily into chondrocytes, adipocytes, and osteocytes. Recent provocative data suggest that they can also express phenotypic characteristics of endothelial, neural, smooth muscle, skeletal myoblasts, and cardiac myocytes. The multilineage potential, no immune reaction if self-transplantation, and the relative easiness of expansion in culture make MSCs a promising source of cells for medical therapies. Recent researches demonstrate that implantation of intramyocardial MSCs can prevent deteriorated remodeling and improve cardiac function, although the underlying mechanisms need to be further clarified. This review summarized recent studies in the recognition of MSCs, particularly the MSC plasticity into cardiomyocytes, factors and signal pathways that mediate MSC differentiation, and animal studies as well as clinic trials of the implantation of MSCs in postinfarct myocardium.

Animals↗
PubMed1989

Inhibition of DNA synthesis causes stem cell differentiation: induction of teratocarcinoma F9 cell differentiation with nucleoside analogues of DNA-synthesis inhibitors and their inducing abilities counterbalanced specifically by normal nucleosides.

Nucleoside analogues inhibiting DNA synthesis can induce cell differentiation in teratocarcinoma cells. We have examined how their abilities to induce F9 cell differentiation were specifically counterbalanced by their corresponding normal nucleosides. We have also compared the differentiation inducing ability of the wild type F9 cells with that of its thymidine kinase-less mutant using plasminogen activator, as a differentiation marker, which is expressed at a very early stage of endodermal cell differentiation and can be assayed quantitatively. The results obtained were clearly explainable by the conventionally accepted action mechanisms of the nucleoside analogues, thus strongly suggesting that their abilities to induce cell differentiation were direct consequences of the inhibition of DNA synthesis; thus this confirms the notion that a close association exists between the inhibition of DNA synthesis and the induction of teratocarcinoma stem cell differentiation.

Animals↗
PubMed2006

Alginate-PLL microencapsulation: effect on the differentiation of embryonic stem cells into hepatocytes.

The emergence of hepatocyte based clinical and pharmaceutical technologies, has been limited by the absence of a stable hepatocyte cell source. Embryonic stem cells may represent a potential solution to this cell source limitation problem since they are highly proliferative, renewable, and pluripotent. Although many investigators have described techniques to effectively differentiate stem cells into a variety of mature cell lineages, their practicality is limited by: (1) low yields of fully differentiated cells, (2) absence of large scale processing considerations, and (3) ineffective downstream enrichment protocols. Thus, a differentiation platform that may be modified to induce and sustain differentiated cell function and scaled to increase differentiated cell yield would improve current stem cell differentiation strategies. Microencapsulation provides a vehicle for the discrete control of key cell culture parameters such as the diffusion of growth factors, metabolites, and wastes. In addition, both cell seeding density and bead composition may be manipulated. In order to assess the feasibility of directing stem cell differentiation via microenvironment regulation, we have developed a murine embryonic stem cell (ES) alginate poly-l-lysine microencapsulation hepatocyte differentiation system. Our results indicate that the alginate microenvironment maintains cell viability, is conducive to ES cell differentiation, and maintains differentiated cellular function. This system may ultimately assist in developing scalable stem cell differentiation strategies.

Albumins↗
PubMed2005

Treatment with stem cell differentiation stage factors of intermediate-advanced hepatocellular carcinoma: an open randomized clinical trial.

There is no standard treatment for patients with advanced hepatocellular carcinoma (HCC). We developed a product containing stem cells differentiation stage factors (SCDSF) that inhibits tumor growth in vivo and in vitro. The aim of this open randomized study was to assess its efficacy in patients with HCC not suitable for resection, transplantation, ablation therapy, or arterial chemoembolization. A total of 179 consecutive patients were enrolled. We randomly assigned the patients to receive either SCDSF or only conservative treatment. Primary end points were tumor response and survival. Secondary end points were performance status and patient tolerance. Randomization was stopped at the second interim analysis (6 months) of the first 32 patients recruited when the inspection detected a significant difference in favor of treatment (p = 0.037). The responses to the therapy obtained in 154 additional patients confirmed previous results. Evaluation of survival showed a significant difference between the group of patients who responded to treatment versus the group with progression of disease (p < 0.001). Of the 23 treated patients with a performance status (PS) of 1, 19 changed to 0. The study indicated the efficacy of SCDSF treatment of the patients with intermediate-advanced HCC.

Aged↗
PubMed2006

Sca-1+ progenitors derived from embryonic stem cells differentiate into endothelial cells capable of vascular repair after arterial injury.

BACKGROUND: Embryonic stem cells possess the ability to differentiate into endothelium. The ability to produce large volumes of endothelium from embryonic stem cells could provide a potential therapeutic modality for vascular injury. We describe an approach that selects endothelial cells using magnetic beads that may be used therapeutically to treat arterial injury. METHODS AND RESULTS: Large numbers of endothelial cells (ECs) with high purity were produced using Sca-1+ cells isolated with magnetic beads from predifferentiated embryonic stem cells (ESCs) cultured in alpha-MEM containing 10 ng/mL VEGF165 for a minimum of 21 days (esEC). The transcription regulator histone deacetylase (HDAC3) was essential for VEGF-induced EC differentiation. Immunofluorescence or fluorescence-activated cell sorter (FACS) analysis revealed that esECs expressed a full range of EC lineage-specific markers including CD31, CD106, CD144, Flk-1, Flt-1, and von Willebrand factor (vWF). FACS analysis confirmed that 99% of esECs were CD31-positive and 75% vWF-positive. Furthermore, almost all cells were positive for DiI-acLDL uptake. When matrigel containing esECs was subcutaneously implanted into mice, various vessel-like structures were observed indicating their endothelial cell like phenotype. In keeping with this, when esECs infected with adenovirus-LacZ were injected into denuded femoral arteries of mice, they were found to form a neo-endothelium that covered the injured areas (86%+/-13.6%), which resulted in a 73% decrease in neointimal area 2 weeks after injury. CONCLUSIONS: We conclude that Sca-1+ cells can differentiate into functional ECs via activation of HDAC3, accelerating re-endothelialization of injured arteries and reducing neointima formation.

Animals↗
PubMed2005

Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation.

Closely related signals often lead to very different cellular outcomes. We found that the differentiation of human mesenchymal stem cells into bone-forming cells is stimulated by epidermal growth factor (EGF) but not platelet-derived growth factor (PDGF). We used mass spectrometry-based proteomics to comprehensively compare proteins that were tyrosine phosphorylated in response to EGF and PDGF and their associated partners. More than 90% of these signaling proteins were used by both ligands, whereas the phosphatidylinositol 3-kinase (PI3K) pathway was exclusively activated by PDGF, implicating it as a possible control point. Indeed, chemical inhibition of PI3K in PDGF-stimulated cells removed the differential effect of the two growth factors, bestowing full differentiation effect onto PDGF. Thus, quantitative proteomics can directly compare entire signaling networks and discover critical differences capable of changing cell fate.

Cell Differentiation↗
PubMed2006

Nuclear and chromatin reorganization in the MHC-Oct3/4 locus at developmental phases of embryonic stem cell differentiation.

Epigenetic gene control is involved in mechanisms of development. Little is known about the cooperation of nuclear and chromatin events in programmed differentiation from mouse embryonic stem cells (ESC). To address this, Oct3/4-positive ESC and differentiated progenies, Sox1-positive neural precursor cells (NPC) and post-mitotic neurons (PMN), were isolated using a stage-selected culture system. We first investigated global nuclear organization at the each stage. Chromocenter preexists in ESC, disperses in NPC and becomes integrated into large heterochromatic foci in PMN, while the formation of PML bodies markedly decreases in neural differentiation. We next focused on the gene-dense MHC-Oct3/4 region. Oct3/4 gene is expressed preferentially adjacent to PML bodies in ESC and are repressed in the absence of chromocenter association in NPC and PMN. Histone deacetylation in NPC, demethylation of lysine 4 of histone H3 (H3K4), tri-methylation of H3K27, and CpG methylation in PMN are targeted for the Oct3/4 promoter within the region. Interestingly, di-methyl H3K4 mark is present in Oct3/4 promoter in NPC as well as ESC. These findings provide insights into the molecular basis of global nuclear reorganization and euchromatic gene silencing in differentiation through the spatiotemporal order of epigenetic controls.

Animals↗