Derivation of Mouse Haploid Trophoblast Stem Cells

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Derivation of Human Trophoblast Stem Cells

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Long Term Culture of Cells Derived from Mouse Blastocysts

The development of mouse blastocysts in primary culture has been followed for up to two months. The trophectoderm layer of the blastocyst gives rise to a monolayer of trophoblast cells; cells resembling both ectoplacental cone cells and primary giant cells are observed. The former can transform to giant cells, presumably secondary trophoblast, after several days in culture. Giant trophoblast cells are evident in the culture for much longer than the normal gestation period. Under the culture conditions described, the proportion of blastocysts showing substantial inner cell mass (ICM) proliferation in vitro is higher than that noted in previous studies. The ICM clumps develop into either egg cylinder-like structures, or, more commonly, into spherical, fluid-filled vesicles. The vesicles, which resemble yolk sac morphologically and biochemically [10, 11], continue to enlarge in size during several weeks of culture. The vesicles are attached to the underlying trophoblast monolayers by a stalk. Cells appear to migrate from this stalk out along the culture dish. The result after two to four weeks of culture is the appearance of a mixed monolayer containing a variety of different cell types. Secondary cultures of blastocyst cells have been continuously maintained in vitro for more than one year. Four lines of cells, all developing from the same pool of blastocysts, have been monitored for morphological, growth and biochemical properties, as well as chromosome number. Each line contained two or more morphologically distinct cell types, clearly indicated by cloning studies after eight months of culture. Doubling times and saturation densities among the four lines differed, as did biochemical properties. Although none of the cell lines resembled trophoblast biochemically after 7.5 months in culture, one line, MB4, possessed a number of biochemical properties in common with midgestation yolk sac. After a further five months of culture, some enzymes in the four lines were relatively unchanged; in other cases, notably with alkaline phosphatase, a sharp drop in enzyme activity was observed. One cell line, MB2, and specifically one of the cell types in this line, produced a yellow-orange pigment with a spectrum resembling that of a heme protein. After 7.5 months of culture, two of the four lines, MB21 and MB31, contained large numbers of cells with a diploid number of chromosomes. However, by 12.5 months in culture, the large majority of metaphases in all four cell lines possessed a hypotetraploid chromosome number. In a number of studies carried out to date, none of the cell lines generated tumors when injected into syngeneic hosts.     

Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells

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Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.     

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.     

Oocyte-like Cells Induced from Mouse Spermatogonial Stem Cells

ABSTRACT: BACKGROUND: During normal development primordial germ cells (PGCs) derived from the epiblast are the precursors of spermatogonia and oogonia. In culture, PGCs can be induced to dedifferentiate to pluripotent embryonic germ (EG) cells in the presence of various growth factors. Several recent studies have now demonstrated that spermatogonial stem cells (SSCs) can also revert back to pluripotency as embryonic stem (ES)-like cells under certain culture conditions. However, the potential dedifferentiation of SSCs into PGCs or the potential generation of oocytes from SSCs has not been demonstrated before. RESULTS: We report that mouse male SSCs can be converted into oocyte-like cells in culture. These SSCs-derived oocytes (SSC-Oocs) were similar in size to normal mouse mature oocytes. They expressed oocyte-specific markers and give rise to embryos through parthenogenesis. Interestingly, the Y- and X-linked testis-specific genes in these SSC-Oocs were significantly down-regulated or turned off, while oocyte-specific X-linked genes were activated. The gene expression profile appeared to switch to that of the oocyte across the X chromosome. Furthermore, these oocyte-like cells lost paternal imprinting but acquired maternal imprinting. CONCLUSIONS: Our data demonstrate that SSCs might maintain the potential to be reprogrammed into oocytes with corresponding epigenetic reversals. This study provides not only further evidence for the remarkable plasticity of SSCs but also a potential system for dissecting molecular and epigenetic regulations in germ cell fate determination and imprinting establishment during gametogenesis.     

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.     

Derivation of Hair-Inducing Cell from Human Pluripotent Stem Cells

Dermal Papillae (DP) is a unique population of mesenchymal cells that was shown to regulate hair follicle formation and growth cycle. During development most DP cells are derived from mesoderm, however, functionally equivalent DP cells of cephalic hairs originate from Neural Crest (NC). Here we directed human embryonic stem cells (hESCs) to generate first NC cells and then hair-inducing DP-like cells in culture. We showed that hESC-derived DP-like cells (hESC-DPs) express markers typically found in adult human DP cells (e.g. p-75, nestin, versican, SMA, alkaline phosphatase) and are able to induce hair follicle formation when transplanted under the skin of immunodeficient NUDE mice. Engineered to express GFP, hESC-derived DP-like cells incorporate into DP of newly formed hair follicles and express appropriate markers. We demonstrated that BMP signaling is critical for hESC-DP derivation since BMP inhibitor dorsomorphin completely eliminated hair-inducing activity from hESC-DP cultures. DP cells were proposed as the cell-based treatment for hair loss diseases. Unfortunately human DP cells are not suitable for this purpose because they cannot be obtained in necessary amounts and rapidly loose their ability to induce hair follicle formation when cultured. In this context derivation of functional hESC-DP cells capable of inducing a robust hair growth for the first time shown here can become an important finding for the biomedical science.     

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.     

Derivation and Characterization of Hepatic Progenitor Cells from Human Embryonic Stem Cells

The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell–derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.     

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye^1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment⁴; however the cells are still capable of forming the different cell types found within the neural retina^1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury⁵, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent^6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation^9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.     

Derivation of Germline Competent Rat Embryonic Stem Cells from DA Rats

The laboratory rat was one of the earliest mammalian species for scientific research and used as animal disease models in physiology,toxicology,behavior,immunology,and tumor-biology for over 150 years (Jacob,1999).However,rat lags far behind mouse in generating human disease models and functional genomic studies because of the lack of authentic rat embryonic stem (ES) cells (Voigt and Serikawa,2009),whereas the first mouse ES cell line was established in 1981 (Evans and Kaufman,1981).By combining two or three kinase inhibitors which target GSK3,MEK and FGF signaling pathways in serum-free N2B27 medium,germline competent rat ES cells were first derived in 2008 (Buehr et al.,2008;Li et al.,2008).     

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos. Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF). Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days. The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state. Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.Download video file.^{(316M, mp4)}     

Isolation of ES-Like Lines of Common Voles of the Genus Microtus from Blastocysts and Germ Cells and as a Result of Fusion of Somatic Cells with Mouse Embryonic Stem Cells

Three and four independent cell lines with limited pluripotency were obtained from the inner cell mass cells of blastocysts and primordial germ cells of common voles, respectively. The results of cytogenetic analysis suggest that all these lines originated from the embryos of F₁ Microtus rossiaemeridionalis × M. arvalis males and had a great number of near-triploid cells already during the early passages. The cells of these lines, like those of the inner cell mass, were characterized by the alkaline phosphatase activity. Nine independent cell lines were obtained as a result of hybridization of the mouse embryonic stem cells and vole splenocytes: eight lines and one line from hybridization with the M. kirgisorum and M. rossiaemeridionalis splenocytes, respectively. The cells of these lines expressed some properties of embryonic stem lines had a chromosome complement similar to the sum of two initial diploid sets of the mouse and vole.