Human embryonic stem cells. Problems and perspectives

Establishment of human embryonic stem cell lines is one the major achievements in the biological science in the XX century and has excited a wide scientific and social response as embryonic stem cells can be regarded in future as unlimited source of transplantation materials for the replacement cell therapy. To date human embryonic cell lines are obtained in more than 20 countries. In our country the embryonic stem cell researches are carried out in the Institute of Cytology RAS and the Institute of Gene Biology RAS. ESC lines are derived from placed in culture inner cell mass of human preimplantation blastocysts used in the in vitro fertilization procedure. Studies with human ESC go in several directions. Much attention is paid to the elaboration of the optimal conditions for ESC cultivation, mainly to the development of cultivation methods excluding animal feeder cells and other components of animal origin. Another direction is a scale analysis of gene expression specific for the embryonic state of the cells and corresponding signaling pathways. Many efforts are concentrated to find conditions for the directed differentiation of ESC into different tissue-specific cells. It has been shown that ESC are able to differentiate in vitro practically into any somatic cells. Some works are initiated to develop methods for the "therapeutic cloning", that is transfer and reactivation of somatic nuclei into enucleated oocytes or embryonic stem cell cytoblasts. Of great importance is human ESC line standardization. However, the standard requirements for the cells projected for research or therapeutic purposes may be different. It has been found that many permanent human ESC lines undergo genetic and epigenetic changes and, therefore, the cell line genetic stability should be periodically verified. The main aim of the review presented is a detailed consideration of the works analyzing the genetic stability of human and mouse ESC lines. Human ESC lines established in our and as well as in other countries couldn't be used so far in clinical practice. It is highly probable that undifferentiated ESC cannot be applied for therapeutic purposes because of the risk of their malignant transformation. Therefore, main efforts should be focused on the production of progenitor and highly differentiated cells suitable for transplantation derived from ESC.     

Human embryonic stem cells: Problems and perspectives

Generation of human embryonic stem cell lines is one of the most important achievements in biological science in the 20th century. It has excited a wide scientific and social response, as embryonic stem cells (ESC) may, in the future, be regarded as an unlimited source of transplantation materials for replacement cell therapy. ESC lines are derived, cultured, inner cell mass from human blastocysts is used in the in vitro fertilization procedure. To date, human embryonic cell lines have been obtained in more than 20 countries. In our country, embryonic stem cell research is carried out in the Institute of Cytology, Russian Academy of Sciences and the Institute of Gene Biology, Russian Academy of Sciences. Studies with human ESC go in several directions. Much attention is paid to finding the most optimal conditions for ESC cultivation, mainly to the development of cultivation techniques excluding animal feeder cells and other components of animal origin. Another direction is a large-scale analysis of gene expression specific to the embryonic state of cells and the corresponding signaling pathways. Great efforts are being focused on the directed differentiation of ESC into various tissue-specific cells. It has been shown that in vitro ESC are able to differentiate into virtually any somatic cells. Works are in progress to develop methods for “therapeutic cloning,” i.e. the transfer of somatic nuclei into enucleated oocytes or embryonic stem cell cytoblasts and their reactivation. Of great importance is the standardization of the human ESC lines. However, standard requirements for cells utilized for research or therapeutic purposes may be different. It has been found that many permanent human ESC lines underwent genetic and epigenetic variations. Therefore, the cell line genetic stability should be periodically verified. The main purpose of the review is to provide a detailed consideration of research on the genetic stability of human and mouse ESC lines. Human ESC lines established both in our country and others could not thus far be used in clinical practice. It is highly probable that undifferentiated ESCs cannot be applied for therapeutic purposes, as there is a risk of their malignant transformation. Therefore, main efforts should be focused on the production ESC progenitor and highly differentiated cells suitable for transplantation.     

Flow cytometric sorting and analysis of human epidermal stem cell candidates

ESC (epidermal stem cells) play a central role in the regeneration of human epidermis. These cells are also responsible for wound healing and neoplasm formation. Efficient isolation of ESC allows their use in medicine and pharmacy as well as in basic science. Cultured keratinocytes and ESC may be used as biological dressing in burn injuries, chronic wounds and hereditary disorders. Therefore, the isolation and characterization of ESC have been goals in biomedical science. Here, we present a flow cytometric method for the isolation and analysis of human ESC candidates. The strategy presented for the isolation of ESC combines previously proposed enzymatic digestion and FACS‐sorting of the obtained cell suspension that utilizes morphological features, integrin‐β1 expression and Rh123 (Rhodamine 123) accumulation of the cells. We also performed a flow cytometric analysis of sorted cells using a cell tracer.     

The effects of dietary phytoestrogens on aromatase activity in human endometrial stromal cells

Dietary phytoestrogens have been reported to inhibit aromatase activity in placental microsomes, but the effects in the human endometrium are unknown. Aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, has recently been shown to be expressed in the endometrium of women with endometriosis and is thought to play a role in the pathophysiology of this disease. Therefore, the objective of this study was to screen dietary phytoestrogens for their ability to inhibit aromatase activity in human endometrial stromal cells (ESC) and identify potential novel therapeutic agents for the treatment of endometriosis. The inhibition of aromatase activity by direct interaction with the dietary phytoestrogens genistein, daidzein, chrysin, and naringenin was tested in a cell free assay. Furthermore, test compound effects on aromatase activity in ESC cultures were also examined. Genistein and daidzein were inactive in the human recombinant aromatase assay whereas naringenin and chrysin inhibited aromatase activity. However, genistein (1 nM to 1 mM) stimulated aromatase activity in ESC whereas other phytoestrogens had no effect. Immunopositive aromatase cells were demonstrated in genistein-treated ESC but not in untreated control cultures. Taken together, our data suggest that genistein can increase aromatase activity in ESC likely via increased enzyme expression.     

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.     

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.     

The expression and possible roles of chemokine CXCL11 and its receptor CXCR3 in the human endometrium

IFN-gamma secreted by a human embryo and trophoblast cells during implantation is suggested to play an important role in implantation and pregnancy. In the present study, we explored expression and possible functions of CXCL11, a CXC chemokine strongly induced by IFN-gamma, and its receptor CXCR3 in the human endometrium. Secreted CXCL11 protein was not detected in cultured endometrial stromal cells (ESC) but was detected in cultured endometrial epithelial cells (EEC). IFN-gamma stimulated the protein levels of CXCL11 in a dose-dependent manner in EEC and ESC. CXCL11 secreted from EEC with 100 ng/ml IFN-gamma was 220-fold of the control, and 100-fold as compared with that secreted from ESC with the same dose of IFN-gamma. CXCR3 was expressed in EEC, ESC, and trophoblast cells. Addition of IFN-gamma to EEC increased the chemotactic activity of its culture medium to trophoblast cells and T cells, and the effect was suppressed by immunoneutralization with Abs of three CXCR3 ligands, including anti-CXCL11 Ab. CXCL11 significantly increased BrdU incorporation of ESC, which was inhibited by a p42/44 MAPK pathway inhibitor PD98059. In contrast, CXCL11 significantly decreased BrdU incorporation and increased the release of lactate dehydrogenase and the positive staining of annexin V in EEC. These findings suggest that IFN-gamma promotes implantation by stimulating EEC to produce CXCL11, which induces migration of trophoblast cells and T cells, proliferation of ESC, and apoptosis of EEC.     

Effect of steroid treatment of endometrial cells on blastocyst development during co-culture

The objective of this study was to determine if treatment of endometrial cells with progesterone or progesterone plus estradiol would improve the development of bovine embryos to the blastocyst stage during co-culture. After IVF, bovine embryos were cultured with oviduct epithelial cells for 3 d. In Experiment 1 the embryos were cultured with a) oviduct epithelial cells; b) endometrial epithelial cells (EEC); c) EEC with 10 ng/ml progesterone (EEC + P); or d) EEC with 10 ng/ml progesterone and 10 pg/ml estradiol (EEC + PE) for 6 d. In Experiment 2 the embryos were cultured with a) oviduct epithelial cells; b) endometrial stromal cells (ESC); c) ESC with 10 ng/ml progesterone (ESC + P); or d) ESC with 10 ng/ml progesterone and 10 pg/ml estradiol (ESC + PE) for 6 d. Results from Experiment 1 showed that endometrial epithelial cells supported development to the blastocyst stage as effectively as the oviduct cells; however, the size of the blastocysts was smaller for the endometrial cells. There was no effect of steroid hormone treatment on development to the blastocyst stage or on the size of the blastocysts. Results from Experiment 2 showed that stromal cells supported development to the blastocyst stage as effectively as oviduct cells. The hatching rate was lower when the embryos were co-cultured with stromal cells than oviduct epithelial cells; but there was no effect of steroid treatment. These data show that untreated endometrial epithelial cells are as effective as oviduct cells in maintaining embryo development to the blastocyst stage. However, embryo development was not improved by steroid treatment of the cells.     

Cytotoxicity of the anticancer drug doxorubicin for human embryonic stem cells

The cytotoxic effect of the anticancer drug doxorubicin (DR) on human embryonic stem cells (ESCs) C910 and fibroblasts spontaneously differentiated from these cells has been examined. The fibroblasts retained a diploid karyotype. It was found that ESCs are more sensitive to DR than fibroblasts: the DR dose killing 20% of cells was 0.01 and 0.1 μg/mL, respectively. DR induced ESC apoptotic death and reduced both ESC and fibroblast proliferation. DR reversibly inhibited ESC, but not fibroblast, proliferation. Thus, we demonstrated that ESCs and differentiated derivatives thereof are distinguished by sensitivity and response to the genotoxic agent.     

胚胎干细胞起源的探讨

目前胚胎干细胞(ESCs)建系的取材来源包括桑椹胚的卵裂球、囊胚的内细胞团(ICM)、上胚层细胞和原始生殖细胞(PGCs),甚至从新生鼠睾丸细胞也分离得到类ES样细胞系。这就提出了一个问题,什么是ESCs最接近的体内细胞来源。传统观念常常把ESCs等同于ICM细胞,也有学者认为ESCs更象上胚层细胞,而在已知的分子标记基因方面,ESCs所具有的特征更接近体内早期生殖细胞。不清楚ESCs最接近的体内细胞来源,可能是制约许多品系小鼠和大多哺乳类动物建系成功率提高的原因之一。ESCs系与EG细胞系的分离条件不同表明,加强对ESCs多能性维持基因调控研究具有重要意义。本文从ESCs的经典概念及其发展,早期胚胎细胞和生殖细胞发育规律,早期胚胎细胞、早期生殖细胞和ESCs的关系等方面进行综合分析,认为ESCs可能有多种接近的体内细胞来源。进一步应通过对ESCs建系不同的取材细胞和不同品系的ESCs间进行比较研究,以便弄清ESCs的来源和转化机制,为提高不同物种ESCs建系效率提供理论支持。     

Protein kinases predominately expressed in human ES cell lines during differentiation

Capacity of human embryonic stem cells (ESC) for unlimited proliferation and differentiation make them an attractive object in fundamental science and medicine. Little is known about the mechanisms that direct cells to particular differentiation or sustain them in an undifferentiated state. Activation of these mechanisms is determined by gene expression mediated by cascades of signal transduction. Protein kinases are essential components of signal pathways. The study of protein kinases expression in ESC and embryoid bodies facilitates a better understanding of the processes underlying the differentiation stages. We isolated cDNA libraries with fragments of catalytic domains of protein kinases expressed in human ESC and embryoid bodies (EB) of hESM01 and hESM02 cell lines. Using Northern hybridization, we revealed a high level of protein kinases MAK-V in human ESC. Expressions of MAK-V, A-RAF-1, MAPK3, IGF1R, NEK3, and NEK7 in ESC and EB in hESM01 and hESM02 cell lines were compared by the semiquantitative method RT-PCR.     

Embryonic stem (ES) cell-derived cardiomyocytes: A good candidate for cell therapy applications

During the last decade, embryonic stem cells (ESC) have unleashed new avenues in the field of developmental biology and emerged as a potential tool to understand the molecular mechanisms taking place during the process of differentiation from the embryonic stage to adult phenotype. Their uniqueness lies in retaining the capacity of unlimited proliferation and to differentiate into all somatic cells. Together with promising results from rodent models, ESC has raised great hope among for human ESC-based cell replacement therapy. ESC could potentially revolutionize medicine by providing a powerful and renewable cell source capable of replacing or repairing tissues that have been damaged in almost all degenerative diseases such as Parkinson's disease, myocardial infarction (MI) and diabetes. Somatic stem cells are an attractive option to explore for transplantation because they are autologous, but their differentiation potential is very limited. Currently, the major sources of somatic cells used for basic research and clinical trials come from bone marrow. But their widespread acceptability has not been gained because many of the results are confusing and inconsistent. The focus here is on human embryonic stem cells (hESCs), using methods to induce their differentiation to cardiomyocytes in vitro. Their properties in relation to primary human cardiomyocytes and their ability to integrate into host myocardium have been investigated into how they can enhance cardiac function. However, important aspects of stem cell biology and the transplantation process remain unresolved. In summary, this review updates the recent progress of ES cell research in cell therapy, discusses the problems in the practical utility of ESC, and evaluates how far this adjunctive experimental approach can be successful.     

Generation of dopamine neurons from human embryonic stem cells in vitro

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth.     

Derivation of hematopoietic stem cells from murine embryonic stem cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny. Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability. Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types. The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol. HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders. However, many patients that might benefit from HSC therapy lack access to suitable donors. ESC could provide an alternative source of HSC for these patients. The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC. In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation. EBs are then dissociated and infected with retroviral HoxB4. Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days. During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100 s of millions of cells. These cells can then be used to rescue and reconstitute lethally irradiated mice.     

Identification and isolation of embryonic stem cells in reproductive endocrinology: theoretical protocols for conservation of human embryos derived from <Emphasis Type="Italic">in vitro</Emphasis> fertilization

Background  

Embryonic stem cells (ESC) are pluripotent cells obtained from the inner cell mass (ICM) of blastocysts derived from in vitro culture associated with reproductive endocrinology therapy. Human ESCs are regarded as highly significant since they retain the capacity to differentiate into any of approximately 200 unique cell types. Human ESC research is controversial because to acquire such cells, the ICM of human blastocysts must be manipulated in a way that renders embryos nonviable and unsuitable for transfer in utero. Techniques to yield competent ESCs with conservation of source blastocysts would satisfy many objections against ESC research, but at present such approaches remain largely untested.     

A novel feeder-free system for human embryonic stem cells and characterization of their sublines with autogenic and allogenic cultivation

We developed a feeder-free system for human embryonic stem cells (ESCs) based on extracellular matrix protein (ECM) as the substrate. ECM was synthesized by mesenchymal stem cells (SC5-MSC) derived from an original ESC line, SC5. The ECM proteins fibronectin and laminin facilitate ESC growth in the feeder-free system. An important component of this system is a conditioned medium from SC5-MSC cells. Two ESC sublines were obtained: SC5-FF cells were cultured in an autogenic, and SC7-FF in an allogenic, feeder-free system. SC5-FF and SC7-FF underwent more than 300 and 115 population doublings, respectively, and retain a normal diploid karyotype. Histochemical and immunofluorescence assays showed that both sublines express undifferentiated ESC markers—alkaline phosphatase, Oct-4, SSEA-4, and TRA-1-81—as well as multidrug resistance transporter ABCG2. PCR assay revealed that undifferentiated SC5-FF cells, like the original SC5 line, maintained on feeder cells express OCT4 and NANOG genes common for somatic cells and DPPA3/STELLA and DAZL genes common for germ line cells. Expression of these genes was gradually diminished during differentiation of embryoid bodies, whereas expression of genes specific for early differentiated cells increased: GATA4, AFP (extraembryonic and embryonic endoderm), PAX6 (neuroectoderm), and BRY (mesoderm). ESC properties (karyotype structure, average time of population doubling, undifferentiated cell number in population) of the SC5 and SC7 and SC5-FF and SC7-FF sublines derived from original ESCs were not altered. It shows that the feeder-free systems, which are more stable than any feeder systems, maintain key ESC properties and may be recommended for fundamental, biomedical, and pharmacological studies performed with human ESCs.     

Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.     

A mouse and embryonic stem cell derived from a single embryo

Embryonic stem cells (ESCs) are a good material for the study of mammalian development, production of genetically modified animals, and drug discovery because they proliferate infinitely while maintaining a multilineage differentiation potency and a normal karyotype. However, ethical considerations limit the use of human embryos for the establishment of ESCs. Recently, ESCs have been produced from blastomeres divided by biopsy in mice and humans. The method is expected to be less controversial because it does not destroy the embryo. However, no one has yet produced both a pup and an ESC from a single embryo. Here, we describe the production of individual/ESC pairs from each of three embryos out of 20 attempts, and is thus considered efficient. Blastomere-derived ESC could differentiate some types of tissues and contribute to chimera mouse. These results show that each blastomere at two-cell stage possesses pluripotency and separated blastomeres maintain viability to develop to a pup or pluripotent ESC.     

Generation of Induced Pluripotent Stem Cell Lines from Tibetan Miniature Pig

Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model.