Using hESCs to Understand Vasculogenesis Processes

The Role of BMP4 Plays in Regulating Vascular Development

My friend, Dr. Steve Stice and team at UGA have published excellent findings on the process of vasculogenesis: N. L. Boyd, S. K. Dhara, R. Rekaya, E. A. Godbey, K. Hasneen, R. R. Rao, F. D. West III, B. A. Gerwe, S. L. Stice. BMP4 Promotes Formation of Primitive Vascular Networks in Human Embryonic Stem Cell–Derived Embryoid Bodies. Exp Biol Med (Maywood) June 2007 vol. 232 no. 6 833-843.
Highlights:
Human embryonic stem cells (hESC) have the capability to produce all of the cells of the body and have been used as in vitro models to study the molecular signals controlling differentiation and vessel assembly. One such regulatory molecule is bone morphogenetic protein-4 (BMP4), which is required for mesoderm formation and vascular/hematopoietic specification in several species. However, hESC grown in feeder-free conditions and treated with BMP4 differentiate into a cellular phenotype highly expressing a trophoblast gene profile. Therefore, it is unclear what role, if any, BMP4 plays in regulating vascular development in hESC. Here we show in two National Institutes of Health–registered hESC lines (BG02 and WA09) cultured on a 3D substrate of Matrigel in endothelial cell growth medium–2 that the addition of BMP4 (100 ng/ml) for 3 days significantly increases the formation and outgrowth of a network of cells reminiscent of capillary-like structures formed by mature endothelial cells (P < 0.05). Analysis of the expression of 45 genes by quantitative real time–polymerase chain reaction on a low-density array of the entire culture indicates a rapid and significant downregulation of pluripotent and most ectodermal markers with a general upregulation of endoderm, mesoderm, and endothelial markers. Of the genes assayed, BMPR2 and RUNX1 were differentially affected by exposure to BMP4 in both cell lines. Immunocytochemistry indicates the morphological structures formed were negative for the mature endothelial markers CD31 and CD146 as well as the neural marker SOX2, yet positive for the early vascular markers of endothelium (KDR, NESTIN) and smooth muscle cells (α-smooth muscle actin [αSMA]). Together, these data suggest BMP4 can enhance the formation and outgrowth of an immature vascular system.

Figures: Least-squared mean gene expression analysis of BMP4 versus control time course for BGO2 and WA09. BG02 (solid lines) and WA09 (dashed lines) were cultured for 3 days ± BMP4, then for another 5 days in EGM-2 only. Samples were collected on Days 0, 2, 4, 6, and 8; total RNA was extracted, and gene expression was analyzed by qRT-PCR. Least-squared mean comparison was then expressed as x-fold change with respect to Day 0 control. The gene expression time course for (A) OCT4, (B) HEY1, (C) GATA3, (D) NESTIN, (E) SOX2, (F) BMPR2, (G) KDR/Flk1, and (H) CD31/PECAM1 are shown. (BG02: −BMP4 = solid diamond; +BMP4 = solid square; WA09: −BMP4 = open triangle; +BMP4 = open circle; * P < 0.05 for BG02 only; ** P < 0.05 for WA09 only; *** P < 0.05 for both BG02 and WA09).http://ebm.sagepub.com/content/232/6/833.full

Images: Early vascular markers KDR, αSMA, and NESTIN are detected within the network structures and elsewhere. Cell cultures were dual-immunostained to detect co-expression of KDR with αSMA (A–C), KDR with NESTIN (D–F), or αSMA with NESTIN (G–I), and the nucleus was counterstained with DAPI. For each case, three different morphologic regions were represented: the residual EB (A, D, and G), the periphery of the EB (B, E, and H), and the thin network structures (C, F, and I). All images were acquired with a ×40 oil objective, and a Z series stack was projected into a single image. A color figure is available in the online version of the journal.

These findings confirm the use of stem cells and the EB as a model of early development including vasculogenesis (3, 4). Using hESC in this manner can provide insights into the mechanisms regulating the earliest events in human vasculogenesis. An understanding of how the vasculature is formed could also be applied to tissue engineering and angiogenic/ischemic therapies. Note: our Mouse Monoclonal Nestin Antibody is an excellent marker for early vasculogensis of the endothelium.

Converting Human Pluripotent Stem Cells into Nociceptors

Methods for differentiating induced pluripotent stem cells (iPSCs) into specific cell types are a requirement for converting the "promise of iPSCs" into reality. The knowledge derived from this research can be leveraged for high throughput  Drug Discovery and ultimately the development of therapies. I am excited to highlight results recently published by Dr. Lorenz Studer and his team at Memorial Sloan-Ketterling: Stuart M Chambers, Yuchen Qi, Yvonne Mica, Gabsang Lee, Xin-Jun Zhang, Lei Niu, James Bilsland, Lishuang Cao, Edward Stevens, Paul Whiting, Song-Hai Shi, Lorenz Studer. Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nature Biotechnology 30, 715–720 (2012) doi:10.1038/nbt.2249.

Abstract: Considerable progress has been made in identifying signaling pathways that direct the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types, including neurons. However, differentiation of hPSCs with extrinsic factors is a slow, step-wise process, mimicking the protracted timing of human development. Using a small-molecule screen, we identified a combination of five small-molecule pathway inhibitors that yield hPSC-derived neurons at >75% efficiency within 10 d of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors, including tetrodotoxin (TTX)-resistant, SCN10A-dependent sodium currents and response to nociceptive stimuli such as ATP and capsaicin. Neuronal fate acquisition occurs about threefold faster than during in vivo development(1), suggesting that use of small-molecule pathway inhibitors could become a general strategy for accelerating developmental timing in vitro. The quick and high-efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain

Figure – LSB3i Differentiation model. Early LSB inhibits trophectoderm, mesendoderm, and non-neural ectoderm cell fates yielding neuroectoderm. CHIR99021, SU5402 and DAPT induce and accelerate neural crest stem cell identity by day 8 and promote rapid differentiation of the neural crest stem cells to nociceptors expressing peptidergic markers by day 10.

Note: Neuromics' TRPV1 Antibody was used as a marker for mature nociceptors.

Check out Supplementary Data for more. I will continue to post links to methods here and @ Neuromics' Stem Cell Research Reagents.

SOX 17 Antibody that Rocks

Our SOX Antibodies are proving to be excellent Stem Cell Markers.  I would like to highlight a pub that references use of our Sox17  and a variety of other markers:

Pere Santamaria, Ignacio Rodríguez-Pizá, Xavier Clemente-Casares, Jun Yamanouchi, Lola Mulero-Perez, Trond Aasen, Angel Raya1 and Juan Carlos Izpisúa Belmonte. Turning Human Epidermis Into Pancreatic Endoderm. The Review of DIABETIC STUDIES 159 Vol. 7-No. 2-2010. DOI 10.1900/RDS.2010.7.158.

This pub references use of a variety of markers for showing differentiation of Human embryonic stem (hES)and induced pluripotent stem (iPS)into into pancreatic endoderm structures.

Real-time PCR and flow cytometry. A: Changes in the levels of different mRNAs in undifferentiated, and progressively differentiated, hES and iPS cells. A series of plates were cultured, as described in the materials and methods section. These were used for RNA extraction at the end of each differentiation stage, immediately prior to change in culture conditions (days 1, 3, 7, 10, and 13). B: Flow cytometric analysis of Sox17 and FoxA2 expression on cells harvested on day 3.