Differentiation of adult stem cells into smooth muscle for vascular tissue engineering - PubMed (original) (raw)

Differentiation of adult stem cells into smooth muscle for vascular tissue engineering

Lisa J Harris et al. J Surg Res. 2011.

Abstract

Background: Herein we evaluate the potential of adipose-derived stem cells (ASC) to differentiate into smooth muscle cells (SMC) and their potential for use in a tissue-engineered vascular graft.

Materials and methods: We isolated ASC (CD13+29+90+) from the peri-umbilical adipose tissue of patients undergoing vascular surgery, and cultured them in media containing angiotensin II (AngII), sphingosylphosphorylcholine (SPC), or transforming growth factor-beta 1 (TGFβ1) for up to 3 weeks. SMC differentiation was assessed by (1) expression of early (calponin, caldesmon) and late (myosin heavy chain, MHC) SMC markers by RT-PCR, qPCR and Western blot, and (2) contraction upon plating on collagen gel. Differentiated ASCs were seeded onto a vascular graft (decellularized saphenous vein) within a bioreactor, and cell attachment was determined using confocal microscopy.

Results: Prior to differentiation, ASC expressed low levels of all three molecular markers. After culture in each differentiating medium, the extent of up-regulation of calponin, caldesmon, and MHC was variable across all cell lines. After seeding onto collagen gel, ASCs differentiated in SPC and TGFβ1 exhibit contractile properties, similar to smooth muscle cell controls. Differentiated stem cells adhered and proliferated on the vascular graft.

Conclusion: These data suggest that human adipose-derived stem cells (1) exhibit variable expression of SMC molecular markers after differentiation, (2) exhibit a contractile phenotype after differentiation with SPC and TGFβ1, and (3) proliferate on a vascular graft scaffold. Thus, ASCs are potentially useful in the construction of autologous arteries.

Copyright © 2011 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Proliferation of ASC and doubling times

Growth curve over 7 days showing ASC (n=3) cultured in non-differentiating media and with media containing growth factors ANG, SPC and TGFβ1 (error bars omitted due to juxtaposition of lines). All p-values for points on curve are not significant. Similarly, doubling times are not significantly different.

Figure 2

Figure 2. Expression of intermediate and late SMC markers after one week of differentiation

RT-PCR for calponin, caldesmon, and MHC was performed for 4 patient cell lines after differentiation in ANG, SPC or TGFβ1 for one week. Expression of calponin was seen with all 3 differentiating agents in patient 4 only. Up-regulation of caldesmon was seen in patients 1 and 3 only. No MHC expression was seen with any cell lines at one week.

Figure 3

Figure 3. Expression of intermediate and late SMC markers after differentiation for two weeks

RT-PCR for calponin, caldesmon, and MHC was performed in 6 patient cell lines after differentiation for two weeks. Up-regulation of calponin was seen in patient 3 only with TGFβ1. Caldesmon up-regulation was variable with 3 of the 6 patients showing up-regulation. Expression of MHC was seen with SPC in patient 2 and with TGFβ1 in patients 3-5. Patients 1 and 6 did not show MHC expression.

Figure 4

Figure 4. Quantification of SMC marker expression after two weeks of differentiation

Quantitative PCR for calponin, caldesmon, and MHC after ASC differentiation (n=3) for two weeks. Up-regulation of calponin was seen with TGFβ1. No up-regulation of caldesmon was seen by ANG, SPC, or TGFβ1. Up-regulation of MHC was seen with ANG and SPC. P-values were not statistically significant.

Figure 5

Figure 5. Protein expression after three weeks of differentiation

Expression of calponin, caldesmon, and MHC protein by Western blot analysis after differentiation in ANG, SPC, or TGFβ1 for three weeks. Up-regulation of calponin was seen with TGFβ1 in cell line 7 and with all three agents in cell lines 8 and 9. No up-regulation of caldesmon was seen, however caldesmon was expressed at baseline and with all three differentiating agents. No expression of MHC was seen.

Figure 6

Figure 6. Assessment of contraction: Collagen gel contraction assay

A. Assessment of basal contractile tone and stimulation of contraction by KCl using collagen gel lattice contraction assay (n=4). Collagen gels without cells show no evidence of contraction. Some basal contractile tone is exhibited by non-differentiated ASC and stem cells differentiated in ANG, SPC and TGFβ1. The addition of KCl results in increased contraction with ASC differentiated with SPC and TGFβ1, but not ANG when compared to undifferentiated ASC. Human arterial smooth muscle cells were used as a positive control. B. Percent increase in contraction after stimulation with KCl normalized to undifferentiated ASC. Stem cells differentiated in SPC and TGFβ1 show significantly increased contraction (p < 0.05) similar to human arterial SMCs.

Figure 7

Figure 7. Attachment of differentiated ASC to vascular graft scaffold

ASCs differentiated in SPC for two weeks were seeded onto vascular graft scaffold (decellularized human greater saphenous vein) (A) and cultured in bioreactor system (B) for 24 hours. Cell attachment as viewed by confocal microscopy at 40× magnification (C). Similar results were obtained with cells differentiated in ANG and TGFβ1. Vascular graft scaffold as viewed by confocal microscopy prior to seeding (D).

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