Clonogenic multipotent stem cells in human adipose tissue differentiate into functional smooth muscle cells

Abstract

Smooth muscle is a major component of human tissues and is essential for the normal function of a multitude of organs including the intestine, urinary tract and the vascular system. The use of stem cells for cell-based tissue engineering and regeneration strategies represents a promising alternative for smooth muscle repair. For such strategies to succeed, a reliable source of smooth muscle precursor cells must be identified. Adipose tissue provides an abundant source of multipotent cells. In this study, the capacity of processed lipoaspirate (PLA) and adipose-derived stem cells to differentiate into phenotypic and functional smooth muscle cells was evaluated. To induce differentiation, PLA cells were cultured in smooth muscle differentiation medium. Smooth muscle differentiation of PLA cells induced genetic expression of all smooth muscle markers and further confirmed by increased protein expression of smooth muscle cell-specific alpha actin (ASMA), calponin, caldesmon, SM22, myosin heavy chain (MHC), and smoothelin. Clonal studies of adipose derived multipotent cells demonstrated differentiation of these cells into smooth muscle cells in addition to trilineage differentiation capacity. Importantly, smooth muscle-differentiated cells, but not their precursors, exhibit the functional ability to contract and relax in direct response to pharmacologic agents. In conclusion, adipose-derived cells have the potential to differentiate into functional smooth muscle cells and, thus, adipose tissue can be a useful source of cells for treatment of injured tissues where smooth muscle plays an important role.

Fig. 1.

PLA cells acquire SMC morphology. Contrast micrographs (×100) are shown. (A) Typical morphology of PLA cells cultured in CM for 4 weeks, which grow in a monolayer. (B) In the presence of SMIM, PLA cells show the typical “hills and valleys” growth pattern of SMC.

Fig. 2.

Induction of SMC markers when PLA cells are grown in smooth muscle differentiation conditions. (A) Induction of gene expression of smooth muscle markers when PLA are exposed to smooth muscle differentiation conditions for 3 and 6 weeks as determined by RT-PCR. (B) Induction of gene expression of all smooth muscle markers as determined by semiquantitative RT-PCR using GAPDH as a control gene. (C) Increases in the expression of ASMA, calponin, and SM22 were confirmed with real-time PCR.

Fig. 3.

Induction of smooth muscle marker protein expression when PLA cells are cultured in SMIM. (A) There is an increase in the expression of ASMA, calponin, caldesmon, and MHC in PLA cells cultured in SMIM compared to cells grown in CM, as determined by immunohistochemistry (×100). (B) Increase in expression of ASMA and induction of MHC confirmed by immunofluorescence (×200). (C) Increase in ASMA and induction of MHC expression confirmed by Western blot analysis.

Fig. 4.

Clonal adipose-derived cells maintain multipotent capacity. (A) When exposed to adipogenic, osteogenic, or leiomyogenic differentiation conditions, ASC clones maintain adipose (Oil red O), osteogenic (alkaline phosphatase), and leiomyogenic (ASMA) phenotype (×100). (B) Representative clone showing new expression of caldesmon and MHC when grown in SMIM as determined by RT-PCR. (C) Representative clone showing increased ASMA, calponin, and SM22 expression when grown in SMIM compared to clone grown in CM as determined by real-time PCR.

Fig. 5.

Smooth muscle-differentiated PLA cells have a response to carbachol that is similar to that seen for SMC. (A) Collagen gels embedded with smooth muscle-differentiated PLA cells contract to carbachol similar to rBSMC. Collagen gels are released at time 0. Only gels with rBSMC and smooth muscle-differentiated PLA cells have contraction after release. Gels are allowed to stabilize for 45 min, at which point 10⁻⁵ M carbachol is added. There is further contraction in rBSMC and smooth muscle-differentiated PLA cells but no response in blank gels or gels embedded with T cells. (B) Smooth muscle-differentiated PLA cells respond to carbachol in a dose-dependent manner. Gels are released at time 0 and allowed to stabilize, and varying doses of carbachol (none, 10⁻¹¹ M, 10⁻⁸ M, and 10⁻⁵ M) are added at 45 min. (C) Atropine blocks carbachol response. Collagen gels embedded with smooth muscle-differentiated PLA cells were released at time 0 and allowed to stabilize. 10⁻⁴ M atropine was added at 45 min, and 10⁻⁵ M carbachol was added at 90 min (open diamonds). The response to carbachol was attenuated compared to a similar experiment where the gel was exposed only to carbachol 10⁻⁵ M at 45 min (filled diamonds).