Paracrine and epigenetic control of trophectoderm differentiation from human embryonic stem cells: the role of bone morphogenic protein 4 and histone deacetylases.

Our understanding of paracrine and epigenetic control of trophectoderm (TE) differentiation is limited by available models of preimplantation human development. Simple, defined media for selective TE differentiation of human embryonic stem cells (hESCs) were developed, enabling mechanistic studies of early placental development. Paracrine requirements of preimplantation human development were evaluated with hESCs by measuring lineage-specific transcription factor expression levels in single cells and morphological transformation in response to selected paracrine and epigenetic modulators. Bone morphogenic protein 4 (BMP4) addition to feeder-free pluripotent stem cells on matrigel frequently formed CDX2-positive TE. However, BMP4 or activin A inhibition alone also produced a mix of mesoderm and extraembryonic endoderm under these conditions. Further, BMP4 failed to form TE from adherent hESC maintained in standard feeder-dependent monolayers. Given that the efficiency and selectivity of BMP4-induced TE depended on medium components, we developed a basal medium containing insulin and heparin. In this medium, BMP4 induction of TE was dose dependent and with activin A inhibition by SB431542 (SB), approached 100% of cells. This paracrine stimulation of pluripotent cells transformed colony morphology from a cuboidal to squamous epithelium quantitatively on day 3, and produced significant multinucleated syncytiotrophoblasts by day 8. Addition of trichostatin A, a histone deacetylase (HDAC) inhibitor, reduced HDAC3, histone H3K9 methylation, and slowed differentiation in a dose-dependent manner. Modulators of BMP4- or HDAC-dependent signaling might adversely influence the timing and viability of early blastocyst developed in vitro. Since blastocyst development is synchronized to uterine receptivity, epigenetic regulators of TE differentiation might adversely affect implantation in vivo.

Enhanced differentiation of embryonic and neural stem cells to neuronal fates on laminin peptides doped polypyrrole.

PPy is a conducting polymer material that has been widely investigated for biomedical applications. hESCs and adult rNSCs were grown on four PPy surfaces doped with PSS or peptide from laminin (p20, p31, and a mixture of p20 and p31) respectively. After 7 d, both PPy/p20 and PPy/p31 promoted neuroectoderm formation from hESCs. After 14 d of culture, surfaces containing p20 showed the highest percentage of neuronal differentiation from hESC, while the PPy/p31 surface showed better cell attachment and spreading. In rNSCs cultures, a higher percentage of neurons were found on the PPy/p20 surface than other surfaces at 7 and 14 d. For differentiated neurons, p20 promoted both the primary and total neurite outgrowth. Longer primary neurites were found on p20-containing surfaces and a longer total neurite length was found on PPy/p20 surface. These results demonstrated that, by doping PPy with different bioactive peptides, differentiation of stem cells seeded at different stages of development is affected.

In vitro neural differentiation of human embryonic stem cells using a low-density mouse embryonic fibroblast feeder protocol.

Human embryonic stem cells (hESCs) have the capacity to self-renew and to differentiate into all components of the embryonic germ layers (ectoderm, mesoderm, endoderm) and subsequently all cell types that comprise human tissues. HESCs can potentially provide an extraordinary source of cells for tissue engineering and great insight into early embryonic development. Much attention has been given to the possibility that hESCs and their derivatives may someday play major roles in the study of the development, disease therapeutics, and repair of injuries to the central and peripheral nervous systems. This tantalizing promise will be realized only when we understand fundamental biological questions about stem cell growth and development into distinct tissue types. In vitro, differentiation of hESCs into neurons proceeds as a multistep process that in many ways recapitulates development of embryonic neurons. We have found in vitro conditions that promote differentiation of stem cells into neuronal precursor or neuronal progenitor cells. Specifically, we have investigated the ability of two federally approved hESC lines, HSF-6 and H7, to form embryonic and mature neuronal cells in culture. Undifferentiated hESCs stain positively for markers of undifferentiated/pluripotent hESCs including surface glycoproteins, SSEA-3 and 4, and transcription factors Oct-3/4 and Nanog. Using reduced numbers of mouse embryonic fibroblasts as feeder substrates, these markers of pluripotency are lost quickly and replaced by primarily neuroglial phenotypes with only a few cells representing other embryonic germ layer types remaining. Within the first 2 weeks of co-culture with reduced MEFs, the undifferentiated hESCs show progression from neuroectodermal to neural stem cell to maturing and migrating neurons to mature neurons in a stepwise fashion that is dependent on both the type of hESCs and the density of MEFs. In this chapter, we provide the methods for culturing pluripotent hESCs and MEFs, differentiating hESCs using reduced density MEFs, and phenotypic analyses of this culture system.

Physical plasticity of the nucleus in stem cell differentiation.

Cell differentiation in embryogenesis involves extensive changes in gene expression structural reorganization within the nucleus, including chromatin condensation and nucleoprotein immobilization. We hypothesized that nuclei in naive stem cells would therefore prove to be physically plastic and also more pliable than nuclei in differentiated cells. Micromanipulation methods indeed show that nuclei in human embryonic stem cells are highly deformable and stiffen 6-fold through terminal differentiation, and that nuclei in human adult stem cells possess an intermediate stiffness and deform irreversibly. Because the nucleo-skeletal component Lamin A/C is not expressed in either type of stem cell, we knocked down Lamin A/C in human epithelial cells and measured a deformability similar to that of adult hematopoietic stem cells. Rheologically, lamin-deficient states prove to be the most fluid-like, especially within the first approximately 10 sec of deformation. Nuclear distortions that persist longer than this are irreversible, and fluorescence-imaged microdeformation with photobleaching confirms that chromatin indeed flows, distends, and reorganizes while the lamina stretches. The rheological character of the nucleus is thus set largely by nucleoplasm/chromatin, whereas the extent of deformation is modulated by the lamina.

Human embryonic stem cells (HSF-6) show greater proliferation and apoptoses when grown on glioblastoma cells than mouse embryonic fibroblasts at day 19 in culture: comparison of proliferation, survival, and neural differentiation on two different feeder cell types.

Phenotypic guidance of embryonic stem (ES) cell fate is paramount if these cells are to be used for tissue repair and regeneration. Our objective was to compare two different cell culture feeders and their effect on proliferation, apoptosis, and differentiation of human (h) ES cells. HSF-6 hES cells were grown in Knockout Dulbecco's modified Eagle medium (DMEM) on mouse embryonic fibro-blasts (MEFs) or U87 glioblastoma cells at densities of 50,000, 100,000, and 150,000 cells/well of a six-well plate for 7, 12, and 19 days. Immunocytochemistry was performed for bromodeoxyuridine (BrdU), TUNEL, and neural differentiation markers including class III beta-tubulin, NeuN, nestin, and doublecortin. Slides were examined by laser confocal microscopy with semiquantitative analyses of marker expression. BrdUand TUNEL-positive cells were primarily, but not exclusively, at edges and between established colonies. BrdU expression was higher on U87 feeders at low and intermediate densities at day 19. Both feeders demonstrated higher BrdU expression at day 7 compared to days 12 and 19. U87 produced more TUNEL-positive cells than MEFs with increasing numbers with increasing density and time in culture. Nuclear Oct-4 staining was seen only at day 7. MEFs appeared to promote greater neural differentiation of hES cells than U87. We conclude hES cells grown on U87 feeders demonstrate greater numbers of apoptotic cells and BrdU-positive cells at day 19. Independent of the feeders, proliferation and apoptosis may be positively correlated. We speculate differences in proliferation, apoptosis, and neural differentiation may be due to differential elaboration of specific cytokines by MEFs and U87.

Lamin A/C expression is a marker of mouse and human embryonic stem cell differentiation.

Nuclear lamins comprise the nuclear lamina, a scaffold-like structure that lines the inner nuclear membrane. B-type lamins are present in almost all cell types, but A-type lamins are expressed predominantly in differentiated cells, suggesting a role in maintenance of the differentiated state. Previous studies have shown that lamin A/C is not expressed during mouse development before day 9, nor in undifferentiated mouse embryonic carcinoma cells. To further investigate the role of lamins in cell phenotype maintenance and differentiation, we examined lamin expression in undifferentiated mouse and human embryonic stem (ES) cells. Wide-field and confocal immunofluorescence microscopy and semiquantitative reverse transcription-polymerase chain reaction analysis revealed that undifferentiated mouse and human ES cells express lamins B1 and B2 but not lamin A/C. Mouse ES cells display high levels of lamins B1 and B2 localized both at the nuclear periphery and throughout the nucleoplasm, but in human ES cells, B1 and B2 expression is dimmer and localized primarily at the nuclear periphery. Lamin A/C expression is activated during human ES cell differentiation before downregulation of the pluripotency marker Oct-3/4 but not before the downregulation of the pluripotency markers Tra-1-60, Tra-1-81, and SSEA-4. Our results identify the absence of A-type lamin expression as a novel marker for undifferentiated ES cells and further support a role for nuclear lamins in cell maintenance and differentiation.