hPS Cell Research Articles

Signals from the Surface to Control Cell Fate Decisions

Human pluripotent stem cells (human embryonic stem cells and induced pluripotent stem cells) have the ability to self‐renew indefinitely and differentiate into all cell types. The repertoire of signals that have been used to date to direct their propagation or differentiation is limited. Additionally, undefined culture conditions are typically used, which complicates determining the critical signaling pathways for expansion or differentiation. Our goal is to identify chemically defined conditions to control signaling pathways to elicit human pluripotent stem (hPS) cell selfrenewal or differentiation. In physiological settings, the microenvironment of the niche transmits information through three types of components: soluble molecules, neighboring cells, and the extracellular matrix. While the focus has been on identifying soluble components that influence cell decisions, insoluble components are likely to have critical roles in orchestrating signaling. To mine this molecular space, we devised arrays of chemically defined surfaces composed of self‐assembled monolayers. From these arrays, we identified surfaces with surprising assets: They not only permit human pluripotent stem cells to differentiate to specific lineages but even instruct them to do so. Our findings highlight the dramatic effects of insoluble cues on stem cell pluripotency and differentiation. Moreover, they underscore the utility of defined surfaces to dissect critical signaling pathways. This research was supported by the NIH (R01‐ GM049975 and R01‐AI055258).

Identification of Human Embryonic Progenitor Cell Targeting Peptides Using Phage Display

Human pluripotent stem (hPS) cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS) cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES) cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken together these data suggest that selection of phage display libraries against a clonal progenitor stem cell population can be used to identify progenitor stem cell targeting peptides. The peptides may be useful for monitoring hPS cell differentiation and for the development of cell enrichment procedures to improve the efficiency of directed differentiation toward clinically relevant human cell types.

Transactivation of ErbB1 and ErbB2 receptors by angiotensin II in normal human prostate stromal cells.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized primarily in the stromal compartment of the human prostate and may regulate stromal as well as epithelial cell growth and survival. The primary cognate HB-EGF receptor, ErbB1, has been shown recently to be transactivated by G-protein coupled receptors (GPCRs) through regulated proteolytic cleavage of the membrane-bound, precursor form of HB-EGF. Previous studies have demonstrated that human prostate tissue, especially tissue from benign prostatic hyperplasia (BPH), has high angiotensin converting enzyme activity, and a high density of angiotensin (Ang) receptors in periurethral stromal cells. Because the pressor peptide Ang II signals through GPCRs, we tested the possibility that Ang II could transactivate ErbB1/ErbB2 in human prostate stromal (hPS) cells. Primary and early passage hPS cells were used as an in vitro model. Cells were stimulated by recombinant HB-EGF or Ang II and total cell lysates were harvested for immunoprecipitation and Western blot. Cell growth was measured by [(3)H]thymidine incorporation assay and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide (MTT) assay. Ang II receptors AT1 and AT2 were expressed in hPS cells. ErbB1 and ErbB2 receptors were activated by HB-EGF. Furthermore, Ang II was able to transactivate both ErbB1 and ErbB2, and this transactivation activity could be abolished by pretreatment with [Glu-52]-diphtheria toxin/CRM197, a specific inhibitor of HB-EGF bioactivity. Consistent with its transactivation activity, Ang II modestly promoted hPS cell growth and this effect was abolished by pretreatment with the ErbB1 antagonist AG1478. These experiments suggest a regulatory role for Ang II in the prostate stroma and implicate the endogenous stromal growth factor HB-EGF as a mediator of Ang II signaling in the prostate.

Anti-proliferative effect of the kinase inhibitor K252a on human prostatic carcinoma cell lines.

Members of the K252 family of kinase inhibitors have been demonstrated to inhibit a number of neurotrophin-mediated cellular responses, and to preferentially inhibit the activity of neurotrophin receptors. In this study, we examined the effects of K252a and K252b on the growth of human prostate carcinoma cells, whose growth is, in part, mediated by a prostatic nerve growth factor(NGF)-like protein(s). K252a inhibited [3H]thymidine incorporation by three androgen-independent prostate tumor cell lines (TSU-pr1, DU-145, and PC-3), under basal growth conditions, and in response to growth stimulation by human prostatic stromal (hPS) cell proteins and serum. K252b, which does not readily penetrate cell membranes, had no significant effect on [3H]thymidine incorporation by the prostate tumor cell lines. The decrease in [3H]thymidine incorporation by the cell lines in response to K252a did not appear to be the result of K252a cytotoxicity at concentrations as high as 100 nM, as measured by the Trypan blue assay for cell viability. Treatment of cells for 25 hours with 100 nM K252a resulted in accumulation of TSU-pr1, DU-145, and PC-3 cells in G0/G1, concurrent with a substantial decrease in cells synthesizing DNA. Treatment of androgen-responsive LNCaP prostatic carcinoma cells for 25 hours with 100 nM K252a also resulted in a significant decrease in DNA synthesis. Human recombinant NGF-mediated phosphorylation of a 140-kDa Trk NGF receptor in the TSU-pr1 cell line was inhibited by treatment with 100 nM K252a. Hence, K252a inhibition of Trk phosphorylation most probably contributed, in part, to the inhibition of prostate tumor cell growth in vitro. These results suggest that the mechanism of K252a action may be useful in the design of potential therapies for prostate cancer treatment.

A sex difference in the progestin receptor system of guinea pig brain.

Female guinea pigs are more sensitive than males to the lordosis-promoting effects of sequential estradiol-progesterone treatment. This study explored a possible cellular basis for this reduced sensitivity. Cytoplasmic progestin receptor concentrations were determined using a [3H]R5020 binding assay for a dissection of brain tissue which included the hypothalamus, preoptic area and septum (HPS) from male and female guinea pigs 40 h after administration of estradiol benzoate (EB). Male guinea pigs injected with 10 micrograms EB have a progestin binder in cytosol from HPS and cerebral cortex that is similar to that in females on the basis of apparent dissociation constant and steroid specificity. Injection of either 1.6 or 10 micrograms EB caused a larger increase in the concentration of cytoplasmic progestin binding in female HPS compared to the same dose in males. A 10 microgram EB injection induced a concentration of progestin binding in male hypothalamus not significantly different from that induced in female hypothalamus by a 1.6 microgram EB injection (a dose that consistently facilitates sexual recepivity in females when followed 40 h later by 0.5 mg progesterone). Despite the induction by EB of a behaviorally-sufficient concentration of cytoplasmic progestin receptors, fewer males (3/10) than females (8/10) responded to EB (10 micrograms)-progesterone treatment with the expression of lordosis. Finally, using a nuclear progestin receptor [3H]R5020 exchange assay, we found that male guinea pigs injected with 10 micrograms EB accumulated a slightly lower concentration of progestin receptors in HPS cell nuclei 4 h after a 0.5 mg progesterone injection than similarly-treated females. Therefore, male guinea pigs show decreased induction of cytoplasmic progestin receptors in HPS after estradiol injection, and a decreased cell nuclear accumulation of these receptors after progesterone injection compared to females. We conclude that this sex difference in the progestin receptor system is insufficient to account, in itself, for the sex difference in behavioral sensitivity to estradiol and progesterone.