Embryonic Stem Cell-like Cell Research Articles

Embryonic Stem Cell-Like Population in Dupuytren's Disease Expresses Components of the Renin-Angiotensin System.

3,3-Diaminobenzidine (DAB) and immunofluorescent immunohistochemical (IHC) staining for (pro)renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) was performed on 4-μm thick formalin-fixed paraffin-embedded sections of DD cords and nodules from 6 patients. Western blotting (WB) and NanoString mRNA analysis were performed to confirm RAS protein expression and transcriptional activation, respectively. IHC staining demonstrated the expression of PRR, ACE, ATIIR1, and ATIIR2 on the ERG+ and CD34+ endothelium of the micro vessels surrounding the DD cords and nodules. PRR was also expressed on the pericyte layer of these microvessels. WB confirmed protein expression of PRR, ACE, and ATIIR2 but not ATIIR1. NanoString analysis confirmed transcriptional activation of PRR, ACE, ATIIR1, but ATIIR2 was below detectable levels. We demonstrated expression of PRR, ATIIR1, ATIIR2, and ACE on the embryonic stem cell-like cell population on the microvessels surrounding DD nodules and cords by IHC staining, although the expression of ATIIR1 was not confirmed by WB and that of ATIIR2 was below detectable levels on NanoString analysis. These findings suggest the embryonic stem cell-like cell population as a potential therapeutic target for DD, by using RAS modulators.

Origins of the breast milk-derived cells; an endeavor to find the cell sources.

Fresh human breast milk consists of a heterogeneous population of cells that may offer a non-invasive source of cells for therapeutic proposes. The aims of this study were to characterize the breast milk-derived cells cultured in vitro. To do this, the cells from human breast milk were cultured and the expression of the CD markers along with the embryonic stem cell markers, endothelial and luminal mammary epithelial cell markers was evaluated by flow cytometry and immunofluorescence. The presence of fetal microchimerism among the isolated cells was also determined by the presence of SRY gene. They were also differentiated into adipocytes and osteoblasts. The results showed that a remarkable number of cells expressed the mesenchymal stem cell (MSC) markers such as CD90, CD44, CD271, and CD146. A subpopulation of the human breast milk-derived cells (HBMDC) also expressed the embryonic stem cell markers, such as TRA 60-1, Oct4, Nanog and Sox2 but not SSEA1 or 4. The frequencies of the cells which expressed the endothelial, hematopoietic cell markers were negligible. SRY gene was not detected in the breast milk isolated cells. A subpopulation of the cells also expressed cytokeratin 18, the marker of luminal mammary epithelial cells. These cells showed the capability to differentiate into adipocytes and osteoblasts. In conclusion, these finding highlighted the presence of cells with various sources in the breast milk. Different stem cells including MSCs or embryonic stem cell-like cell along with the exfoliated cells from luminal epithelial cells were found among the isolated cells. The breast milk-derived stem cells might be considered as a non-invasive source of the stem cells for therapeutic purpose.

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

Derivation of goat embryonic stem cell-like cell lines from in vitro produced parthenogenetic blastocysts

Parthenogenesis is the biological phenomenon by which embryonic development is initiated without male contribution. Parthenogenesis is a very useful method of derivation of embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. The aim of the present study was to derive and characterize goat embryonic stem cell-like cells from in vitro developed blastocysts following parthenogenetic activation of goat oocytes. Two parthenogenetic embryonic stem cell-like cell (gPESC) lines were established. The gPES cell-like cell colonies showed typical ESC morphology and expressed ESC specific markers such as alkaline phosphatase, Oct-4, Sox-2, Nanog, TRA-1-60, SSEA-4 and TRA-1-81. Genetic stability of the cell lines was proven to be preserved via karyotype analysis which showed normal karyotype. In suspension culture in absence of feeder layer and without LIF, the embryonic stem cell-like cells produced embryoid bodies and following prolonged culture differentiated into several types of cells including neuron like, epithelium like and cardiac muscle cells. Cell specific markers were observed all cells expressed positive marker genes. All of these results demonstrated the feasibility to isolate and establish goat parthenogenetic ES cell-like cell lines, which provides an important tool for studying epigenetic effects in ESCs and developmental biology as well as gene targeting to produce genetically modified livestock.

INTRAMYOCARDIAL INJECTION OF INDUCED PLURIPOTENT STEM CELLS IMPROVES LEFT VENTRICULAR FUNCTION AND PERFUSION: A PRECLINICAL STUDY IN A PORCINE MODEL OF ACUTE MYOCARDIAL INFARCTION

ObjectivesInduced pluripotent stem (iPS) cells, a novel embryonic stem cell-like pluripotent stem cell, have potential to differentiate into different cardiovascular cells and repair the injured heart. The potential therapeutic benefits...

Progresses and Challenges in Optimization of Human Pluripotent Stem Cell Culture

The pressing demand to elucidate the biology of human embryonic stem (ES) cells and to realize their therapeutic potential has greatly promoted the progresses in the optimization of the culture systems used for this highly promising cell type. These progresses include the characterization of exogenous regulators of pluripotency and differentiation, the development of animal-free, defined, and scalable culture systems, and some pioneering efforts to establish good manufactory practice facilities to derive and expand clinical-grade human ES cells and their derivatives. All of these advancements appear to be also applicable to the derivation and culture of human induced pluripotent stem cells, an ES cell-like cell type derived from somatic cells via reprogramming. This review attempts to summarize these progresses and discuss some of the remaining challenges.

Somatic cell dedifferentiation/reprogramming for regenerative medicine.

The concept of dedifferentiation or reprogramming of a somatic cell into a pluripotent embryonic stem cell-like cell (ES-like cell), which give rise to three germ layers and differentiate various cell types, opens a new era in stem cell biology and provides potential therapeutic modality in regenerative medicine. Here, we outline current dedifferentiation/reprogramming methods and their technical hurdles, and the safety and therapeutic applications of reprogrammed pluripotent stem cells in regenerative medicine. This review summarizes the concept and data of somatic cell nuclear transfer, fusion of somatic cells with ES cells, viral or non-viral transduction of pluripotency-related genes into somatic cells, introduction of extract (or proteins) of pluripotent cells into somatic cells. Dedifferentiated/reprogrammed ES-like cells could be a perfect genetic match (autologous or tailored pluripotent stem cells) for future applications. Further studies regarding technical refinements as well as mechanistic analysis of dedifferentiation induction and re-differentiation into specific cell types will provide us with the substantial application of pluripotent stem cells to therapeutic purposes.

Development of a Pluripotent ES-like Cell Line from Asian Sea Bass (Lates calcarifer)—An Oviparous Stem Cell Line Mimicking Viviparous ES Cells

We report a pluripotent embryonic stem cell-like cell line designated as SBES from blastula stage embryos of Asian sea bass (Lates calcarifer), which is an economically important cultivable and edible marine fish species in India. The SBES cells were cultured at 28 degrees C in Leibovitz L-15 medium supplemented with 20% fetal bovine serum without a feeder layer. The ES-like cells were round or polygonal and grew exponentially in culture. The SBES cells exhibited an intense alkaline phosphatase activity and expression of transcription factor Oct 4. The undifferentiated state of these cells was maintained at low seeding densities and the cells formed embryoid bodies when seeded in bacteriological plates. On treatment with all-trans retinoic acid, these cells differentiated into neuron-like cells, muscle cells, and beating cardiomyocytes, indicating their pluripotency. This embryonic ES-like cell line derived from an oviparous fish blastula conserved several peculiar features of viviparous mammalian embryonic stem cell lines. The present study highlights the importance and potential of piscine ES-like cell line for stem cell research without evoking ethical issues and invasive interventions sparing mammalian embryos.

279 COMPARISON OF DIFFERENT METHODS OF ISOLATION OF EMBRYONIC STEM CELL-LIKE CELLS FROM IN VITRO-PRODUCED GOAT BLASTOCYSTS

The present study was carried out to compare different isolation procedures for embryonic stem (ES) cell-like cells from in vitro-produced goat blastocysts. Goat oocytes were aspirated from slaughterhouse-derived ovaries; matured in vitro in maturation medium containing TCM-199 (HEPES modified) with 5 µg mL–1 FSH, 10 µg mL–1 LH, 1 µg mL–1 estradiol-17β, BSA, and 10% estrous goat serum; and fertilized by freshly collected buck semen. The fertilized oocytes were cultured in embryo development medium containing TCM-199 (HEPES modified), 50 µg mL–1 sodium pyruvate, 3.5 µg mL–1 L-glutamine, gentamicin, essential amino acids, nonessential amino acids, BSA, and 10% fetal calf serum, along with goat oviductal cells for further development. A total of 250 blastocysts and 50 hatched blastocysts were used to isolate and culture goat ES cell-like cells. Inner cell mass cells (ICMs) were isolated mechanically from 100 blastocysts and 20 hatched blastocysts and enzymatically from 50 blastocysts and 15 hatched blastocysts. The ICMs were cultured on 10 µg mL–1 mitomycin-C-inactivated goat fetal fibroblast feeder layer. Primary colony formation was significantly higher (P < 0.01) when hatched blastocysts were used for ICM isolation (28/35; 80%) than when blastocysts were used (77/150; 51.3%). However, when ICMs were isolated mechanically, the percentage of primary colony formation was significantly higher (P < 0.01) for blastocysts (66/100; 66%) as well as for hatched blastocysts (18/20; 90%) than when ICMs were isolated enzymatically (15/50; 30%, and 11/15; 73%), respectively, for blastocysts and hatched blastocysts. ES cell-like cells were also isolated from intact blastocysts and intact hatched blastocysts. But the primary colony formation (27/100; 27%), and (6/15; 40%), respectively, was significantly low (P < 0.01) compared to that for mechanically isolated ICMs. Statistical analysis was done by chi-squire test. When the putative ES cell-like cells were subcultured enzymatically (0.25% trypsin–EDTA), they differentiated after 3 passages. However, when the mechanical subculture procedure was followed, the ES cell-like cells maintained their pluripotent state up to the 9th passage, and three goat ES cell-like cell lines were produced (gES-1, gES-2, and gES-3). The putative ES cell-like cells were stained with alkaline phosphatase substrate solution (Sigma, St. Louis, MO, USA). The RT-PCR mediated expression of Oct-4 was done by a special kit Cells-to-cDNA™ II (Ambion, Austin, TX, USA). The ES cell-like cells expressed both alkaline phosphatase and Oct-4. It may be concluded that mechanical isolation of hatched goat blastocysts is the best method for isolation of goat embryonic stem cell-like cells, and they are positive for alkaline phosphatase and Oct-4.

Isolation and characterization of embryonic stem cell‐like cells from in vitro‐produced buffalo (Bubalus bubalis) embryos

This study was carried out to isolate and characterize buffalo embryonic stem (ES) cell-like cells from in vitro-produced embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 120 blastocysts whereas 28 morulae were used for the isolation of blastomeres mechanically. The ICM cells/ blastomeres were cultured on mitomycin-C-treated feeder layer. Primary cell colony formation was higher (P < 0.05) for hatched blastocysts (73.1%, 30/41) than that for early/expanded blastocysts (25.3%, 20/79). However, no primary cell colonies were formed when blastomeres obtained from morulae were cultured. Primary colonies were formed in 14.1% (12/85) of intact blastocyst culture, which was significantly lower (P < 0.05) than that of 41.6% for ICM culture. These colonies were separated by enzymatic or mechanical disaggregation. Using mechanical disaggregation method, the cells remained undifferentiated and two buffalo ES cell-like cell lines (bES1, bES2) continued to grow in culture up to eight passages. However, disassociation through enzymatic method resulted in differentiation. Undifferentiated cells exhibited stem cell morphological features, normal chromosomal morphology, and expressed specific markers such as alkaline phosphatase (AP) and Oct-4. Cells formed embryoid bodies (EBs) in suspension culture; extended culture of EBs resulted in formation of cystic EBs. Following prolonged in vitro culture, these cells differentiated into several types of cells including neuron-like and epithelium-like cells. Furthermore, the vitrified-thawed ES cell-like cells also exhibited typical stem cell characteristics. In conclusion, buffalo ES cell-like cells could be isolated from in vitro-produced blastocysts and maintained in vitro for prolonged periods of time.

Long-term culture and differentiation of rat embryonic stem cell-like cells into neuronal, glial, endothelial, and hepatic lineages.

The in vitro differentiation of mouse embryonic stem cells into different somatic cell types such as neurons, endothelial cells, or myocytes is a well-established procedure. Long-term culture of rat embryonic stem cells is known to be hazardous, and attempts to differentiate these cells in vitro so far have been unsuccessful. We herein describe stable long-term culture of an alkaline phosphatase-positive rat embryonic stem cell-like cell line (RESC) and its differentiation into neuronal, endothelial, and hepatic lineages. RESCs were characterized by typical growth in single cells as well as in embryoid bodies when cultured in the presence of leukemia inhibitory factor. RESC expressed stage-specific-embryonic antigen-1 and the major histocompatibility complex class I molecule. For neuronal differentiation, cells were incubated with medium containing 10(-6) M retinoic acid for 14 days. For endothelial differentiation, RESCs were grown on Matrigel for 14 days, and for induction of hepatocyte-specific antigen expression, RESCs were grown in medium supplemented with fibroblast growth factor-4. Differentiated cells exhibited typical morphological changes and expressed neuronal (nestin, mitogen-activated protein-2, synaptophysin), glial (S100, glial fibrillary acid protein), endothelial (panendothelial antibody, CD31) and hepatocyte-specific (alpha-fetoprotein [alphaFP], albumin, alpha-1-antitrypsin, CK18) antigens. In addition, expression of hepatocyte-specific genes (alphaFP, transthyretin, carbamoyl-phosphate synthetase, and coagulation factor-2) was detected by reverse transcription polymerase chain reaction. We were able to culture RESCs under stable, long-term conditions and to initiate programmed differentiation of RESCs to endothelial, neuronal, glial, and hepatic lineages in the rat species.

Establishment of Embryonic Stem (ES) Cell-Like Cell Lines Derived from Bovine Blastocysts Obtained by In Vitro Culture of Oocytes Matured and Fertilized In Vitro.

Inner cell masses (ICM) were isolated immunosurgically from 110 bovine blastocysts derived from in vitro produced day-8 embryos and subsequently cultured in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum, 10-4 M mercaptoethanol, nonessential amino acids and nucleosides on mitomycin C-treated mouse embryonic fibroblast feeder cells. Approximately 5 days after plating, growing ICMs were dissociated by trypsinization and the colonies composed of cells with ES cell-like morphology were selected for further passage at 4 to 6 days intervals. Sixty seven percent of the ICMs were clumped and 7.8% of the ICMs brought into culture produced ES cell-like cell lines. Histochemical staining revealed that all cell lines after five passages were positive for the alkaline phosphatase activity. Immunohistochemical localization of the carbohydrate antigen by 4C9 monoclonal antibody reactivity also gave a positive result similar to that seen immediately following immunodissection. Cytogenetic analysis revealed a karyotype with an euploid number of chromosomes in 48.1 to 71.0% of cells. In all cell lines, dissociated cells formed embryoid bodies 8 to 10 days after culture in the absence of feeder cells. These results suggest that the bovine ES cell-like cell lines established were pluripotent, at least in vitro.

Bovine embryonic stem cell-like cell lines cultured over several passages.

A total of 14 microsurgically produced zona pellucida-free bovine demi-blastocysts were cultured for 3 days in tissue culture medium (TCM) 199 supplemented with 10% heat-inactivated newborn calf serum (NBCS). Developing embryos were continuously cultured in TCM 199 plus 10% NBCS on a feeder-layer of murine embryonic fibroblasts, that had been incubated with mitomycin C (10 μg/ml) for 3 h prior to the onset of embryo cultivation to block mitotic activity of the fibroblasts. After 2 days, 3 expanded blastocysts were attached to the feeder-layer and both trophoblastic cells and inner cell mass (ICM) cells became apparent on the 9th day of culture in 2 out of the 3 expanded blastocysts. Five days later, the ICM cells were disaggregated by a short-term trypsin treatment. The resulting dissociated clumps were seeded on a new murine embryonic fibroblast feeder-layer and covered with modified minimum essential medium (MEM)-Alpha with 10% fetal calf serum (FCS), 0.1 mm mercaptoethanol, 4.5 g/l glucose and 20 mM HEPES-buffer (=passage 0). To prevent differentiation of the cells, approximately 1/3 of the MEM-Alpha was replaced by MEM previously incubated on cell line 5637 containing leucaemia inhibitory factor (LIF) for 3 days. Colonies of embryonic stem cell (ES)-like cells were observed 5 days after the 1st passage. These colonies were repeatedly passaged at approximately 2-week intervals. Two bovine ES-like cell lines were established, which grew considerably slower than murine ES cells, but were lost after the 4th passage, possibly because of toxic effects of a new FCS batch. After cytogenetic analysis, 16 out of 18 metaphase plates contained an euploid number of chromosomes with 2 X-chromosomes and 58 autosomes. Distribution of G-banding on the chromosomes of ES-like cells was in accordance with the diploid set of the bovine genome. ES-like cells were fused to in vitro matured bovine oocytes and, upon successful fusion, cultured in vitro over 5 days. Successful fusion was observed in 79.8% (67/84), 31.3% initiated cleavege and 10.4% reached the 8-16 cell stage at termination of culture.