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The Murine Bladder Supports a Population of Stromal Sca-1+/CD34+/lin- Mesenchymal Stem Cells.

Lilly MA, Kulkulka NA, Firmiss PR, Ross MJ, Flum AS, Santos GB, Bowen DK, Dettman RW, Gong EM - PLoS ONE (2015)

Bottom Line: These cells function normally during organ homeostasis, but become dysregulated after organ injury.These cells differentiated into other mesenchymal lineages (chondrocytes, adipocytes and osteocytes) upon culture in induction medium.Partial obstruction resulted in upregulation of fibrosis genes within the Sca-1+/CD34+/lin- population.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology, Stanley Manne Children's Research Institute, Anne and Robert H. Lurie Children's Hospital of Chicago, 225 E. Chicago Ave. Box 225, Chicago, Illinois, 60611, United States of America.

ABSTRACT
Bladder fibrosis is an undesired end point of injury of obstruction and often renders the smooth muscle layer noncompliant. In many cases, the long-term effect of bladder fibrosis is renal failure. Despite our understanding of the progression of this disease, little is known about the cellular mechanisms that lead to a remodeled bladder wall. Resident stem (progenitor) cells have been identified in various organs such as the brain, heart and lung. These cells function normally during organ homeostasis, but become dysregulated after organ injury. Here, we aimed to characterize a mesenchymal progenitor cell population as a first step in understanding its role in bladder fibrosis. Using fluorescence activated cell sorting (FACS), we identified a Sca-1+/ CD34+/ lin- (PECAM-: CD45-: Ter119-) population in the adult murine bladder. These cells were localized to the stromal layer of the adult bladder and appeared by postnatal day 1. Cultured Sca-1+/ CD34+/ lin- bladder cells self-renewed, formed colonies and spontaneously differentiated into cells expressing smooth muscle genes. These cells differentiated into other mesenchymal lineages (chondrocytes, adipocytes and osteocytes) upon culture in induction medium. Both acute and partial obstruction of the bladder reduced expression of CD34 and changed localization of Sca-1 to the urothelium. Partial obstruction resulted in upregulation of fibrosis genes within the Sca-1+/CD34+/lin- population. Our data indicate a resident, mesenchymal stem cell population in the bladder that is altered by bladder obstruction. These findings provide new information about the cellular changes in the bladder that may be associated with bladder fibrosis.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0141437.g004: 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.

Mentions: We cultured small numbers of Sca-1+/CD34+/lin- cells in non-induction medium and periodically fixed them for immunofluorescence analysis (Fig 4). We used antibodies against mature smooth muscle cytoskeletal proteins (SMM and calponin) and SRF, a transcription factor expressed early in smooth muscle development [25]. After 24h and 48h, the majority of sorted cells expressed Sca-1 and SRF, but not SMM or calponin, (Fig 4A, 4B, 4E, 4F, 4I and 4J). After 4d, Sca-1+/SMM+, Sca-1-/SMM+, Sca-1+/calponin+ and Sca-1-/calponin+ cells were observed in colonies, suggesting that the cells that were initially sorted were dividing and daughters were becoming cells that expressed SMM and calponin (Fig 4C, 4G, 4R and 4S). After 7d, while some Sca-1+ cells remained (Fig 4L, arrow), most cells in colonies were positive for SMM, calponin and SRF (Fig 4D, 4H and 4L). qPCR analysis of Sca-1+/CD34+/lin- cells at the time of sort versus after 7 days in α-MEM culture was consistent with our findings in immunofluorescence analysis (Fig 4M–4Q). After 7d in culture, Sca-1 expression decreased by approximately 1.2-fold and CD34 expression decreased by over 250-fold (Fig 4M and 4N). A 5-fold increase in ACTA2 and an 18-fold increase SMM (Myh11) expression was observed at 7d in culture. (Fig 4P and 4Q). SRF levels decreased by about half after 7d in culture (Fig 4O and 4T). These observations, combined with our immunofluorescence analysis supported the hypothesis that Sca-1+/CD34+/lin- cells acquire properties of smooth muscle cells when cultured in non-induction medium.


The Murine Bladder Supports a Population of Stromal Sca-1+/CD34+/lin- Mesenchymal Stem Cells.

Lilly MA, Kulkulka NA, Firmiss PR, Ross MJ, Flum AS, Santos GB, Bowen DK, Dettman RW, Gong EM - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4634995&req=5

pone.0141437.g004: 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.
Mentions: We cultured small numbers of Sca-1+/CD34+/lin- cells in non-induction medium and periodically fixed them for immunofluorescence analysis (Fig 4). We used antibodies against mature smooth muscle cytoskeletal proteins (SMM and calponin) and SRF, a transcription factor expressed early in smooth muscle development [25]. After 24h and 48h, the majority of sorted cells expressed Sca-1 and SRF, but not SMM or calponin, (Fig 4A, 4B, 4E, 4F, 4I and 4J). After 4d, Sca-1+/SMM+, Sca-1-/SMM+, Sca-1+/calponin+ and Sca-1-/calponin+ cells were observed in colonies, suggesting that the cells that were initially sorted were dividing and daughters were becoming cells that expressed SMM and calponin (Fig 4C, 4G, 4R and 4S). After 7d, while some Sca-1+ cells remained (Fig 4L, arrow), most cells in colonies were positive for SMM, calponin and SRF (Fig 4D, 4H and 4L). qPCR analysis of Sca-1+/CD34+/lin- cells at the time of sort versus after 7 days in α-MEM culture was consistent with our findings in immunofluorescence analysis (Fig 4M–4Q). After 7d in culture, Sca-1 expression decreased by approximately 1.2-fold and CD34 expression decreased by over 250-fold (Fig 4M and 4N). A 5-fold increase in ACTA2 and an 18-fold increase SMM (Myh11) expression was observed at 7d in culture. (Fig 4P and 4Q). SRF levels decreased by about half after 7d in culture (Fig 4O and 4T). These observations, combined with our immunofluorescence analysis supported the hypothesis that Sca-1+/CD34+/lin- cells acquire properties of smooth muscle cells when cultured in non-induction medium.

Bottom Line: These cells function normally during organ homeostasis, but become dysregulated after organ injury.These cells differentiated into other mesenchymal lineages (chondrocytes, adipocytes and osteocytes) upon culture in induction medium.Partial obstruction resulted in upregulation of fibrosis genes within the Sca-1+/CD34+/lin- population.

View Article: PubMed Central - PubMed

Affiliation: Developmental Biology, Stanley Manne Children's Research Institute, Anne and Robert H. Lurie Children's Hospital of Chicago, 225 E. Chicago Ave. Box 225, Chicago, Illinois, 60611, United States of America.

ABSTRACT
Bladder fibrosis is an undesired end point of injury of obstruction and often renders the smooth muscle layer noncompliant. In many cases, the long-term effect of bladder fibrosis is renal failure. Despite our understanding of the progression of this disease, little is known about the cellular mechanisms that lead to a remodeled bladder wall. Resident stem (progenitor) cells have been identified in various organs such as the brain, heart and lung. These cells function normally during organ homeostasis, but become dysregulated after organ injury. Here, we aimed to characterize a mesenchymal progenitor cell population as a first step in understanding its role in bladder fibrosis. Using fluorescence activated cell sorting (FACS), we identified a Sca-1+/ CD34+/ lin- (PECAM-: CD45-: Ter119-) population in the adult murine bladder. These cells were localized to the stromal layer of the adult bladder and appeared by postnatal day 1. Cultured Sca-1+/ CD34+/ lin- bladder cells self-renewed, formed colonies and spontaneously differentiated into cells expressing smooth muscle genes. These cells differentiated into other mesenchymal lineages (chondrocytes, adipocytes and osteocytes) upon culture in induction medium. Both acute and partial obstruction of the bladder reduced expression of CD34 and changed localization of Sca-1 to the urothelium. Partial obstruction resulted in upregulation of fibrosis genes within the Sca-1+/CD34+/lin- population. Our data indicate a resident, mesenchymal stem cell population in the bladder that is altered by bladder obstruction. These findings provide new information about the cellular changes in the bladder that may be associated with bladder fibrosis.

No MeSH data available.


Related in: MedlinePlus