Sunday, July 28, 2013

eSC Derived hNP1 Neural Progenitors Astrocytic Differentiation

Protocol for Driving hNP1TM Human Neural Progenitors to Astrocytes

There is a great demand for an easy way to generate human astrocytes in culture. I am pleased to present a protocol for differentiating our hNP1 Cells to Astrocytes. This comes from my friend Dr. Steve Stice and his team at ArunA Biomedical and University of Georgia: Majumder A, Dhara SK, Swetenburg R, Mithani M, Cao K, Medrzycki M, Fan Y, Stice SL. Inhibition of DNA methyltransferases and histone deacetylases induces astrocytic differentiation of neural progenitors. Stem Cell Res. 2013 Jul;11(1):574-86. doi: 10.1016/j.scr.2013.03.003. Epub 2013 Apr 2.

These enriched non-transformed human astrocyte progenitors will provide a critical cell source to further our understanding of how astrocytes play a pivotal role in neural function and development. Human neural progenitors derived from pluripotent embryonic stem cells and propagated in adherent serum-free cultures provide a fate restricted renewable source for quick production of neural cells; however, such cells are highly refractive to astrocytogenesis and show a strong neurogenic bias, similar to neural progenitors from the early embryonic central nervous system (CNS). We found that several astrocytic genes are hypermethylated in such progenitors potentially preventing generation of astrocytes and leading to the proneuronal fate of these progenitors. However, epigenetic modification by Azacytidine (Aza-C) and Trichostatin A (TSA), with concomitant signaling from BMP2 and LIF in neural progenitor cultures shifts this bias, leading to expression of astrocytic markers as early as 5days of differentiation, with near complete suppression of neuronal differentiation.

Images: Morphology and gene expression after 15 and 30 days of differentiation of cells with astrocytic treatment. Bright field images of hNP cells differentiated (A) with or (B) without astrocytic treatment. A and B compare morphology of cultured cells in treated vs. untreated differentiation at 15 days. Treated and untreated cells were cryopreserved at d6 and subsequently thawed and cultured for an additional 9 days. Flow cytometry analysis to determine percent of GFAP+ and S100B+ cells at d15 of differentiation. Data is presented as histograms for (C) GFAP and (D) S100B with corresponding immunoreactive cells in insets from a parallel culture. Immunocytochemistry detects expression of (E) GFAP with S100B (inset showing distinct staining for both markers), (F) GFAP with GLAST, and (G) GFAP with ALDH1L1 at d30 of differentiation.

The Protocol:  For astrocytic differentiation of hNP cells, neuronal differentiation media were supplemented with BMP2 (20 ng/mL) and combinations of Aza-C and TSA; Aza-C (500 nM), TSA (100 nM) and BMP2 (20 ng/mL) for 2 days, with one complete media change in between, followed by differentiation media supplemented with BMP2 but not with Aza-C or TSA. Cells were harvested prior to analysis at 5, 15 or 30 days of treatment or for cryopreservation at d6 or d10 of differentiation. For cryopreservation, cells were dissociated with Accutase™ and frozen in differentiation media containing10% DMSO. Viability was assessed at 30 days in Aza-C and TSA treated cultures by trypan blue exclusion, and datawas acquired using a Cellometer Auto T4® (Nexcelom Biosciences).

I will keep you updated on new differentiation protocols for our potent, pure and widely used hNP1 Human Neural Progenitors to new phenotypes.

Monday, July 15, 2013

MAP-2-A Versatile Neuron Marker

Neuromics is a leader in providing Neuron-Glial Markers for Neuroscientists.

We are constantly on the search for publications that reference use of these markers in unique applications. In this posting I would like to share a publication where researchers used on of our MAP-2 antibodies to stain medial superior olive (MSO) neurons. Baumann Veronika, Lehnert Simon, Leibold Christian, Koch Ursula. Tonotopic Organization of the Hyperpolarization-activated Current (Ih) in the Mammalian Medial Superior Olive. Front. Neural Circuits 7:117. doi: 10.3389/fncir.2013.00117.
 ...Following recording, slices were fixed in 4% paraformaldehyde for 30 min. After extensive washing in phosphate-buffered saline (PBS) slices were exposed to blocking buffer (0.5% trition X-100/0.1% saponin/1% BSA in PBS) followed by incubation with the primary antibody (chicken anti-microtubule-associated protein 2, MAP2, 1:1000, Neuromics) in blocking buffer. Slices were then rinsed in washing buffer (0.5% Trition X-100/0.1% saponin in PBS) and immunoreactivity was visualized by incubating the slices with the Cy3-conjugated secondary antibody raised in donkey (1:300; Dianova). Finally, slices were washed and mounted on slides with vectashield mounting reagent (Vector Laboratories, USA)...

Here the MAP-2 antibody is used to help identify the dorsal, medial and ventral portion of the MSO of p18 and p22 gerbils.

Figure . Ih varies systematically along the dorsoventral axis. (A) A brain slice containing the MSO with Alexa-488-filled neurons (green) verifies the distribution of the patched neurons along the dorsoventral axis (red: MAP-2). (B) Pharmacologically isolated Ih current traces were elicited by depolarizing and hyperpolarizing voltage steps from −60.5 mV to potentials between −40.5 mV and −120.5 mV for 1 s in 5 mV step increment and then to −100.5 mV for 0.5 s to elicit the tail current to determine the voltage dependence of Ih activation. Current traces are representative for the dorsal, the intermediate and the ventral part of the MSO. (C) I-V relationships of steady-state (red arrow in B) Ih density for ventral (n = 15), intermediate (n = 12) and dorsal (n = 18) neurons emphasize that Ih density amplitudes are smallest in dorsal neurons and largest in ventral neurons (C1). Ih density amplitudes for a voltage step to −110.5 mV (C2). (D) Weighted activation time constants at −110.5 mV (D1). The weighted activation time constants are voltage dependent and largest in the dorsal part of the MSO (D2). (E) The voltage-dependence of Ih activation was measured from the tail current 20 ms after the end of the voltage steps (red arrow) (E1). Values were fitted with a Boltzmann function to obtain the half-maximal activation voltage. In dorsal neurons the Ih activation curve is shifted to more negative voltages (E2). Half-maximal activation voltage was measured in each experiment and averaged (E3). Black symbols: dorsal neurons; gray symbols: intermediate neurons; white symbols: ventral neurons. **P < 0.01, ***P < 0.001, single-factor ANOVA test followed by a Scheffe's post-hoc test.

I will continue to post interesting applications using our Neuron-Glial Markers.

Wednesday, July 03, 2013

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.
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).

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 (34). 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.