Human embryonic stem cell-derived vascular progenitor cells capable of endothelial and smooth muscle cell function

Abstract

Objective: Previous studies have demonstrated development of endothelial cells (ECs) and smooth muscle cells (SMCs) as separate cell lineages derived from human embryonic stem cells (hESCs). We demonstrate CD34(+) cells isolated from differentiated hESCs function as vascular progenitor cells capable of producing both ECs and SMCs. These studies better define the developmental origin and reveal the relationship between these two cell types, as well as provide a more complete biological characterization.

Materials and methods: hESCs are cocultured on M2-10B4 stromal cells or Wnt1-expressing M2-10B4 for 13 to 15 days to generate a CD34(+) cell population. These cells are isolated using a magnetic antibody separation kit and cultured on fibronectin-coated dishes in EC medium. To induce SMC differentiation, culture medium is changed and a morphological and phenotypic change occurs within 24 to 48 hours.

Results: CD34(+) vascular progenitor cells give rise to ECs and SMCs. The two populations express respective cell-specific transcripts and proteins, exhibit intracellular calcium in response to various agonists, and form robust tube-like structures when cocultured in Matrigel. Human umbilical vein endothelial cells cultured under SMC conditions do not exhibit a change in phenotype or genotype. Wnt1-overexpressing stromal cells produced an increased number of progenitor cells.

Conclusions: The ability to generate large numbers of ECs and SMCs from a single vascular progenitor cell population is promising for therapeutic use to treat a variety of diseased and ischemic conditions. The stepwise differentiation outlined here is an efficient, reproducible method with potential for large-scale cultures suitable for clinical applications.

Figure 1. CD34⁺ Vascular Progenitor Cells Capable of Endothelial Cell Differentiation

(A) hESC-derived cells after 15 days of differentiation demonstrating expression of CD34, CD31, and Flk1 both before (presort) and after (postsort) sorting for CD34+ cells. (B) After sorting, the CD34⁺ cells are placed in endothelial cell (EC) culture medium on fibronectin coated tissue culture plastic. After 2–4 passages, expression of EC specific surface markers and lectins can be detected by flow cytometry. Histograms demonstrate red plot as isotype control or corresponding competitive sugar control (for lectins) in each panel, and blue plot is stained for surface antigen or lectin, as indicated. hESC-ECs also exhibited uptake of di-ac-LDL. (C) Immunofluorescent staining of hESC-EC: (left to right) CD31, VE-Cadherin, vWF, eNOS, dilac-LDL. Original magnification 100× for each plot. Blue signal represents DAPI stained nuclei. (D) RT-PCR of hESC-derived ECs: mRNA expression of 7 transcripts for typical EC genes as indicated above each row. (E) Transmission Electron Micrograph (TEM) images of hESC derived ECs cultured under endothelial cell culture conditions show release of microparticles of approximately 100nm in size (as indicated by arrows) from the cell surface.

Figure 2. Micrographic, Immunofluorescent, and Genotypic Assessment of hESC-SMCs

(A) Schema of hESC-EC culture in EGM2 media, then development of hESC-SMCs via change of conditions to DMEM with TGF-β1 and PDGF-BB. Photomicrographs show hESC-ECs with characteristic EC morphology and tube formation on Matrigel. After change to SMC conditions, cells flatten out and show pronounced intracellular fibrils. Genotypic analysis via RTPCR showed specific expression of SMC transcripts. (B) Further phenotypic analysis was conducted via immunofluorescent staining for SMC specific intracellular proteins. Staining of hESC-SMCs (left to right): SM22, alpha-smooth muscle actin (α-SMA), and calponin. Blue signal represents Hoechst 33258 stained nuclei. Original magnification 200×. (C) Staining of hESC-ECs (left to right): SM22, a-SMA, and calponin. Blue signal represents Dapi counter stain. Original magnification 20×. (D) RT-PCR of hESC-derived SMCs for eight common SMCs genes (and β-actin control), as indicated above each indicated lane. To control for contaminating genomic DNA, reactions were also done under conditions with no reverse transcriptase. For expression of EC and SMC genes, all values are means and standard deviations of 3 RT-PCR analysis of independent experiments.

Figure 3. Increased Development of CD34+ Vascular Progenitor Cells From Wnt1 Expressing Stromal Cell Differentiation

Limiting dilution analysis was done to quantify vascular progenitor cells from hESCs allowed to differentiate on M2-10B4 stromal cells that did not over-express Wnt proteins, or M2-10B4 cells that over-expressed either Wnt1 or Wnt5, as indicated. Numerical values shown as progenitor cells per 10,000 cells. Error bars represent Standard Error of the Mean of n= 4 individual experiments; * Wnt1 (p=0.0279) and * Wnt5 (p=0.0309).

Figure 4. Intracellular calcium response of hESC-EC and hESC-SMC to pharmacological agonists

Single cell preparations were exposed to pharmacological agonists as indicated. Responses of each cell type were measured via a fluorimetric ratio using fura-2. (A) hESC-SMC phase image (B) Fura2 loaded hESC-SMCs prior to agonist exposure (C) Fluorometric change post-agonist exposure (oxytocin); pseudocolor scale: low ratios indicated by blue color and high ratios indicated by yellow to red color (A–C original magnification 400×). (D) Representative of time course graph of seven individual hESC-SMCs exposed to oxytocin, ET-1, ATP and Bradykinin. Each line represents the ratios obtained from an individual cell in successive image pairs. (E) Graphic summary comparing responses of 100 undifferentiated hESC, 100 hESC-ECs , and 105 hESC-SMCs to specific agonsits: bradykinin (BK), endothelin-1 (ET-1), oxytocin (Oxy), histamine (Hist), ATP, serotonin (5-HT), vasopressin (AVP), norepinephrine (NE), and carbachol (Carb). Each population tested was comprised of cells from more than one culture. Not all agonists elicited response in all populations.

Figure 5. In-vitro Matrigel Tube Formation Assay

To assess functional potential of hESC-ECs and hESC-SMCs in vitro, cells were cultured on Matrigel in a 50/50 EC/SMC medium. Vascular tube structures formed in both hESC-EC and hESC-EC/hESC-SMC cultures, with the co-cultured cells interacting to form more robust, denser tube structures. (A–C) hESC-ECs alone (D–F) hESC-ECs co-cultured with hESC-SMCs (G) Fluorescent image of GFP-expressing hESC-ECs co-cultured with mCherry-expressing hESC-SMC demonstrate close interaction between the two cell populations in these in vitro Matrigel cultures. Images in (G) show low power (25×) of both cell populations, as well as higher power (200×) of separate GFP and mCherry expressing cells, as well as co-localization of fluorescent cells with phase image. (Original magnifications: A 40×, B 100×, C 400×, D 40×, E 100× F 400×, G 25× and 200×).