Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions

We previously showed that Wnt3a could stimulate human embryonic stem （hES） cell proliferation and affect cell fate determination. In the absence of feeder cell--derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wntl and Wnt5a. While Wntl displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wntl enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wntl gene in immortalized human adult fibroblast （HAFi） cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt tmnsgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells （W3R） that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.

A focus on parietal cells as a renewing cell population

The fact that the acidsecreting parietal cells undergo continuous renewal has been ignored by many gastroenterologists and cell biologists. In the past, it was thought that these cells were static. However, by using 3Hthymidine radioautography in combination with electron microscopy, it was possible to demonstrate that parietal cells belong to a continuously renewing epithelial cell lineage. In the gastric glands, stem cells anchored in the isthmus region are responsible for the production of parietal cells. The stem cells give rise to three main progenitors: prepit, preneck and preparietal cells. Parietal cells develop either directly from the noncycling preparietal cells or less commonly via differentiation of the cycling prepit and preneck cell progenitors. The formation of a parietal cell is a sequential process which involves diminishment of glycocalyx, production of cytoplasmic tubulovesicles, an increase in number and length of microvilli, an increase in number and size of mitochondria, and fi nally, expansion and invagination of the apical membrane with the formation of an intracellular canalicular system. Little is known about the genetic counterparts of these morphological events. However, the time dimension of parietal cell production and the consequences of its alteration on the biological features of the gastric gland are well documented. The production of a new parietal cell takes about 2 d. However, mature parietal cells have a long lifespan during which they migrate bidirectionally while their functional activity for acid secretion gradually diminishes. Following an average lifespan of about 54 d, in mice, old parietal cells undergo degeneration and elimination. Various approaches for genetic alteration of the development of parietal cells have provided evidence in support of their role as governors of the stem/progenitor cell proliferation and differentiation programs. Revealing the dynamic features and the various roles of parietal cells would help in a better understanding of the biological features of the gastric glands and would hopefully help in providing a basis for the development of new strategies for prevention, early detection and/or therapy of various gastric disorders in which parietal cells are involved, such as atrophic gastritis, peptic ulcer disease and gastric cancer.

Cell proliferation during hair cell regeneration induced by Math1 in vestibular epithelia in vitro

Hair cell regeneration is the fundamental method of correcting hearing loss and balance disorders caused by hair cell damage or loss. How to promote hair cell regeneration is a hot focus in current research. In mammals, cochlear hair cells cannot be regenerated and few vestibular hair cells can be renewed through spontaneous regeneration. However, Math1 gene transfer allows a few inner ear cells to be transformed into hair cells in vitro or in vivo. Hair cells can be renewed through two possible means in birds： supporting cell differentiation and transdifferentiation with or without cell division. Hair cell regeneration is strongly associated with cell proliferation. Therefore, this study explored the relationship between Math1-induced vestibular hair cell regeneration and cell division in mammals. The mouse vestibule was isolated to harvest vestibular epithelial cells. Ad-Math1-enhanced green fluorescent protein （EGFP） was used to track cell division during hair cell transformation.5-Bromo-2′-deoxyuridine （BrdU） was added to track cell proliferation at various time points. Immunocytochemistry was utilized to determine cell differentiation and proliferation. Results demonstrated that when epithelial cells were in a higher proliferative stage, more of these cells differentiated into hair cells by Math1 gene transfer. However, in the low proliferation stage, no BrdU-positive cells were seen after Math1 gene transfer. Cell division always occurred before Math1 transfection but not during or after Math1 transfection, when cells were labeled with BrdU before and after Ad-Math1-EGFP transfection. These results confirm that vestibular epithelial cells with high proliferative potential can differentiate into new hair cells by Math1 gene transfer, but this process is independent of cell proliferation.

Crypt region localization of intestinal stem cells in adults

The intestinal epithelial lining plays a central role in the digestion and absorption of nutrients,but exists in a harsh luminal environment that necessitates continual renewal.This renewal process involves epithelial cell proliferation in the crypt base and later cell migration from the crypt base to the luminal surface.This process is dependent on multi-potent progenitor cells,or stem cells,located in each crypt.There are about 4 to 6 stem cells per crypt,and these stem cells are believed to generate distinct end-differentiated epithelial cell types,including absorptive cells,goblet cells,enteroendocrine cells and Paneth cells,while also maintaining their own progenitor cell state.Earlier studies suggested that intestinal stem cells were located either in the crypt base interspersed between the Paneth cells [i.e.crypt base columnar(CBC) cell model] or at an average position of 4 cells from the crypt base [i.e.label-retaining cells(LRC +4) model].Recent studies have employed biomarkers in the in vivo mammalian state to more precisely evaluate the location of these progenitor cells in the intestinal crypt.Most notable of these novel markers are Lgr5,a gene that encodes a G-protein-coupled receptor with expression restricted to CBC cells,and Bmi 1,which encodes a chromatin remodeling protein expressed by LRC.These studies raise the possibility that there may be separate stem cell lines or different states of stem cell activation involved in the renewal of normal mammalian intestinal tract.