The Murine Bladder Supports a Population of Stromal Sca-1+/CD34+/lin- Mesenchymal Stem Cells
Fig 4
Colony forming assays with Sca-1+/CD34+/lin- cells demonstrate that individual cells form colonies and differentiate into cells expressing smooth muscle genes.
(A-D) Confocal micrographs of Sca-1+/CD34+/lin- sorted cells cultured on glass coverslips and stained with antibodies to Sca-1 (green) and SMM (red) at varying time points up to 7 days. Arrow in (C) shows a single Sca-1+ cell next to a group of SMM+ cells (arrowheads) at 4d of culture. (E-H) Confocal micrographs of Sca-1+/CD34+/lin- sorted cells cultured on glass coverslips in α-MEM and stained with antibodies to Sca-1 (green) and calponin (red) at varying time points up to 7 days. (I-L) Confocal micrographs of Sca-1+/CD34+/lin- sorted cells cultured on glass coverslips in α-MEM and stained with antibodies to Sca-1 (green) and SRF (red) at varying time points. Arrow (L) shows a single cell co-expressing SRF and Sca-1 next to a group of SRF+, Sca-1- cells stained in red. (M-Q) qPCR analysis of Sca-1+/CD34+/lin- expression of 5 genes at the time of sort versus after 7 days in α-MEM culture. Sca-1, CD34, and SRF expression levels are normalized to expression levels after 7 d in culture. Myh11 and ACTA2 are normalized to expression levels at the time of sort. Analysis represents two technical replicates of two separate sorts. Each sort consisted of 5 or 6-pooled CD1 mouse bladder cells. Asterisks (N, P, Q) represent significance values of P < 0.05 * and P < 0.01 ** after Student’s T-test. (R, S) Quantification of spontaneous in vitro differentiation Sca-1+/CD34+/lin- cells into SMM and calponin expressing cells. (T) Quantification of in vitro expression of SRF and Sca-1 in Sca-1+/CD34+/lin- cells. (R, S, T) Cells from three independent sorting experiments were fixed at 24h, 48h, 4d and 7d. Coverslips were stained for Sca-1 and either SMM, calponin or SRF. Green lines represent cells stained only with Sca-1. Gold lines represent cells stained with Sca-1 and SMM, Sca-1 and calponin or Sca-1 and SRF. Red lines represent cells stained with SMM, calponin or SRF but not Sca-1. Black lines represent cells that did not stain at all. Error bars (R-T) represent standard error of the mean.