The cellular form of the prion protein guides the differentiation of human embryonic stem cells into neuron-, oligodendrocyte-, and astrocyte-committed lineages

Prion protein, PrP^C, is a glycoprotein that is expressed on the cell surface beginning with the early stages of embryonic stem cell differentiation. Previously, we showed that ectopic expression of PrP^C in human embryonic stem cells (hESCs) triggered differentiation toward endodermal, mesodermal, and ectodermal lineages, whereas silencing of PrP^C suppressed differentiation toward ectodermal but not endodermal or mesodermal lineages. Considering that PrP^C might be involved in controlling the balance between cells of different lineages, the current study was designed to test whether PrP^C controls differentiation of hESCs into cells of neuron-, oligodendrocyte-, and astrocyte-committed lineages. PrP^C was silenced in hESCs cultured under three sets of conditions that were previously shown to induce hESCs differentiation into predominantly neuron-, oligodendrocyte-, and astrocyte-committed lineages. We found that silencing of PrP^C suppressed differentiation toward all three lineages. Similar results were observed in all three protocols, arguing that the effect of PrP^C was independent of differentiation conditions employed. Moreover, switching PrP^C expression during a differentiation time course revealed that silencing PrP^C expression during the very initial stage that corresponds to embryonic bodies has a more significant impact than silencing at later stages of differentiation. The current work illustrates that PrP^C controls differentiation of hESCs toward neuron-, oligodendrocyte-, and astrocyte-committed lineages and is likely involved at the stage of uncommitted neural progenitor cells rather than lineage-committed neural progenitors.     

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.     

Tendon‐derived stem/progenitor cell aging: defective self‐renewal and altered fate

Aging is a major risk factor for tendon injury and impaired tendon healing, but the basis for these relationships remains poorly understood. Here we show that rat tendon‐derived stem/progenitor cells (TSPCs) differ in both self‐renewal and differentiation capability with age. The frequency of TSPCs in tendon tissues of aged animals is markedly reduced based on colony formation assays. Proliferation rate is decreased, cell cycle progression is delayed and cell fate patterns are also altered in aged TSPCs. In particular, expression of tendon lineage marker genes is reduced while adipocytic differentiation increased. Cited2, a multi‐stimuli responsive transactivator involved in cell growth and senescence, is also downregulated in aged TSPCs while CD44, a matrix assembling and organizing protein implicated in tendon healing, is upregulated, suggesting that these genes participate in the control of TSPC function.     

Involvement of neuropeptide Y and its Y1 and Y5 receptors in maintaining self‐renewal and proliferation of human embryonic stem cells

Neuropeptide Y (NPY) and NPY receptors are widely expressed in various organs and cell types and have been shown to have pleiotropic functions. However, their presence or role in human embryonic stem cells (hESCs) remains unknown. We now show that undifferentiated hESCs primarily express NPY and its Y1 and Y5 receptors. Inhibition of NPY signalling using either the selective NPY Y1 or Y5 receptor antagonist reduces the maintenance of self‐renewal and proliferation of undifferentiated hESCs. We also provide compelling evidence that exogenous NPY supports the long‐term growth of undifferentiated hESCs in the absence of feeder cell factors using only knockout serum replacement media. Further, NPY facilitates the use of chemically defined medium made up of N2/B27 supplement and basic fibroblast growth factor (bFGF) for hESC feeder‐free culture. Our results indicate that both Y1 and Y5 receptors appear to be involved in the NPY‐mediated activation of AKT/protein kinase B and extracellular signal‐regulated kinase 1/2 (ERK1/2) in hESCs. Notably, only Y1 receptor, but not Y5 receptor, is responsible for the NPY‐induced activation of cAMP‐response element binding (CREB) in hESCs. These results provide the first evidence that NPY and its Y1 and Y5 receptors have potential role in maintaining hESC self‐renewal and pluripotency. We demonstrate the underlying importance of NPY signalling and its usefulness in the development of a defined and xeno‐free culture condition for the large‐scale propagation of undifferentiated hESCs.     

The cell cycle inhibitor p27Kip1 controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle,Brachyury and Twist

The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27^Kip1 cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27^Kip1 in hESC lead to a G₁ phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27^Kip1 caused an elongated/scatter cell-like phenotype involving upregulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27^Kip1 protein occupies the Twist1 gene promoter and manipulation of p27^Kip1 by gain and loss of function is associated with Twist gene expression changes. These results define p27^Kip1 expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27^Kip1 in controlling an epithelial to mesenchymal transition (EMT) in hESC.     

The cell cycle inhibitor p27Kip1 controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle,Brachyury and Twist

The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27^Kip1 cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27^Kip1 in hESC lead to a G₁ phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27^Kip1 caused an elongated/scatter cell-like phenotype involving upregulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27^Kip1 protein occupies the Twist1 gene promoter and manipulation of p27^Kip1 by gain and loss of function is associated with Twist gene expression changes. These results define p27^Kip1 expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27^Kip1 in controlling an epithelial to mesenchymal transition (EMT) in hESC.     

Smurf2 regulates hematopoietic stem cell self‐renewal and aging

The age‐dependent decline in the self‐renewal capacity of stem cells plays a critical role in aging, but the precise mechanisms underlying this decline are not well understood. By limiting proliferative capacity, senescence is thought to play an important role in age‐dependent decline of stem cell self‐renewal, although direct evidence supporting this hypothesis is largely lacking. We have previously identified the E3 ubiquitin ligase Smurf2 as a critical regulator of senescence. In this study, we found that mice deficient in Smurf2 had an expanded hematopoietic stem cell (HSC) compartment in bone marrow under normal homeostatic conditions, and this expansion was associated with enhanced proliferation and reduced quiescence of HSCs. Surprisingly, increased cycling and reduced quiescence of HSCs in Smurf2‐deficient mice did not lead to premature exhaustion of stem cells. Instead, HSCs in aged Smurf2‐deficient mice had a significantly better repopulating capacity than aged wild‐type HSCs, suggesting that decline in HSC function with age is Smurf2 dependent. Furthermore, Smurf2‐deficient HSCs exhibited elevated long‐term self‐renewal capacity and diminished exhaustion in serial transplantation. As we found that the expression of Smurf2 was increased with age and in response to regenerative stress during serial transplantation, our findings suggest that Smurf2 plays an important role in regulating HSC self‐renewal and aging.     

Fam60a defines a variant Sin3a‐Hdac complex in embryonic stem cells required for self‐renewal

Sin3a is the central scaffold protein of the prototypical Hdac1/2 chromatin repressor complex, crucially required during early embryonic development for the growth of pluripotent cells of the inner cell mass. Here, we compare the composition of the Sin3a‐Hdac complex between pluripotent embryonic stem (ES) and differentiated cells by establishing a method that couples two independent endogenous immunoprecipitations with quantitative mass spectrometry. We define the precise composition of the Sin3a complex in multiple cell types and identify the Fam60a subunit as a key defining feature of a variant Sin3a complex present in ES cells, which also contains Ogt and Tet1. Fam60a binds on H3K4me3‐positive promoters in ES cells, together with Ogt, Tet1 and Sin3a, and is essential to maintain the complex on chromatin. Finally, we show that depletion of Fam60a phenocopies the loss of Sin3a, leading to reduced proliferation, an extended G1‐phase and the deregulation of lineage genes. Taken together, Fam60a is an essential core subunit of a variant Sin3a complex in ES cells that is required to promote rapid proliferation and prevent unscheduled differentiation.     

Inheritance of OCT4 predetermines fate choice in human embryonic stem cells

It is well known that clonal cells can make different fate decisions, but it is unclear whether these decisions are determined during, or before, a cell's own lifetime. Here, we engineered an endogenous fluorescent reporter for the pluripotency factor OCT4 to study the timing of differentiation decisions in human embryonic stem cells. By tracking single‐cell OCT4 levels over multiple cell cycle generations, we found that the decision to differentiate is largely determined before the differentiation stimulus is presented and can be predicted by a cell's preexisting OCT4 signaling patterns. We further quantified how maternal OCT4 levels were transmitted to, and distributed between, daughter cells. As mother cells underwent division, newly established OCT4 levels in daughter cells rapidly became more predictive of final OCT4 expression status. These results imply that the choice between developmental cell fates can be largely predetermined at the time of cell birth through inheritance of a pluripotency factor.     

Neural differentiation of human embryonic stem cells

Availability of human embryonic stem cells (hESC) has enhanced human neural differentiation research. The derivation of neural progenitor (NP) cells from hESC facilitates the interrogation of human embryonic development through the generation of neuronal subtypes and supporting glial cells. These cells will likely lead to novel drug screening and cell therapy uses. This review will discuss the current status of derivation, maintenance and further differentiation of NP cells with special emphasis on the cellular signaling involved in these processes. The derivation process affects the yield and homogeneity of the NP cells. Then when exposed to the correct environmental signaling cues, NP cells can follow a unique and robust temporal cell differentiation process forming numerous phenotypes.     

Mechanisms that mediate stem cell self-renewal and differentiation

Stem cells have two common properties: the capacity for self-renewal and the potential to differentiate into one or more specialized cell types. In general, stem cells can be divided into two broad categories: adult (somatic) stem cells and embryonic stem cells. Recent evidence suggested that tumors may contain "cancer stem cells" with indefinite potential for self-renewal. In this review, we will focus on the molecular mechanisms regulating embryonic stem cell self-renewal and differentiation, and discuss how these mechanisms may be relevant in cancer cells.     

A matter of life and death: self‐renewal in stem cells

If Narcissus could have self‐renewed even once on seeing his own reflection, he would have died a happy man. Stem cells, on the other hand, have an enormous capacity for self‐renewal; in other words, the ability to replicate and generate more of the same. In adult organisms, stem cells reside in specialized niches within each tissue. They replenish tissue cells that are lost during normal homeostasis, and on injury they repair damaged tissue. The ability of a stem cell to self‐renew is governed by the dynamic interaction between the intrinsic proteins it expresses and the extrinsic signals that it receives from the niche microenvironment. Understanding the mechanisms governing when to proliferate and when to differentiate is vital, not only to normal stem cell biology, but also to ageing and cancer. This review focuses on elucidating conceptually, experimentally and mechanistically, our understanding of adult stem cell self‐renewal. We use skin as a paradigm for discussing many of the salient points about this process, but also draw on the knowledge gained from these and other adult stem cell systems to delineate shared underlying principles, as well as highlight mechanistic distinctions among adult tissue stem cells. By doing so, we pinpoint important questions that still await answers.