Contractile responses of smooth muscle cells differentiated from rat neural stem cells

Abstract

To characterize the functional differentiation of neural stem cells into smooth muscle cells, multipotent stem cells in the central nervous system (CNS) were isolated from rat embryonic day 14 (E14) cortex and cultured by neurosphere formation in serum‐free medium in the presence of 10 ng ml−1 of basic fibroblast growth factor. Differentiation was induced by the addition of 10 % fetal bovine serum to low‐density cultures (2.5 × 103 cells cm−2). Immunological analyses and reverse transcriptase‐polymerase chain reaction indicated that the differentiated cells expressed smooth‐muscle‐specific marker proteins such as SM‐1, SM‐2, and SMemb myosin heavy chains, SM‐22, basic calponin and α‐smooth‐muscle actin, but not the astrocyte marker glial fibrillary acidic protein. To examine whether smooth‐muscle‐like cells that are differentiated from CNS stem cells possess the characteristics of contractile smooth muscle, we prepared reconstituted collagen gel fibres and measured their contractile tension. The reconstituted fibres were prepared by thermal gelation of collagen and the differentiated cells. The fibres contracted in response to treatment with KCl (80 mm), ACh (100 μm), endothelin‐1 (10 nm), endothelin‐2 (10 nm), and prostaglandin F2α (100 μm). ACh‐induced contraction was partially inhibited by the L‐type voltage‐dependent Ca2+ channel inhibitor nifedipine and by the intracellular Ca2+ chelator 1,2‐bis (2‐aminophenoxy) ethane‐N,N,N’,N'‐tetraacetic acid acetoxymethyl ester, the myosin light chain kinase inhibitor ML‐9, the Rho kinase inhibitor Y‐27632, dibutyryl cAMP and 8‐bromo‐cGMP. These results suggest that CNS stem cells give rise to smooth muscle cells in vitro that have an identical contractile function to smooth muscle in vivo.

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

“…The ability of SMC to contract in response to a physiological stimulus has been considered to be the main proof that myocytes are in a differentiated state (30,31). In this study, we developed a system to evaluate the ability of cells to respond to a variety pharmacologic agents by tracking the movement of glass beads dispersed in a collagen gel embedded with cells.…”

Angiogenesis In Vitro: Vascular Tube Formation From the Differentiation of Neural Stem Cells

“…In addition to generating a wide variety of neural cells, NSCs are capable of producing cells of other tissues (1,2). We have reported that NSCs give rise to smooth muscle cells in vitro, which have a typical contractile function identical to smooth muscles in vivo (3). Although the significance of the NSC origin of smooth muscle cells in physiological and pathological states needs to be evaluated, these observations suggest that smooth muscle cells may be in part derived from NSCs.…”

“…Total RNA was isolated from the cells (5´10 5 cells) using an Isogen RNA purification kit (Nippon Gene, Tokyo) as deacribed (3). One microgram of total RNA was reverse transcribed using SuperScript II enzyme (Life Technologies Inc., Rockville, MD, USA) and oligo (dT)12-18 primers.…”

“…Immunoperoxidase staining of cultured cells was performed as described previously (3). For double-labeled immunofluorescent staining, primary antibodies developed in two different species were incubated together.…”

Agonist- and depolarization-induced signals for myosin light chain phosphorylation and force generation of cultured vascular smooth muscle cells

“…The collagen gel fibers were kept for one or two additional days in the serumfree culture media. Oishi et al showed that cultured smooth muscle cells in reconstituted fibers exhibited elongated bipolar shapes and were oriented parallel to the longitudinal axis of the fibers (Oishi et al, 2000;Oishi et al, 2002). The rodshaped fibers were cut into 4-mm-long strips.…”