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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

In vitro differentiation of mouse bladder mesenchymal stem cells.(A) Sca-1+/CD34+/lin- cells grown in osteogenic medium, two weeks post-sort, stained with alizarin red for calcium deposition. (B) Quantification of alizarin red by absorbance at 556nm. Red hatched bar represents Sca-1+/CD34+/lin- cells grown in osteogenic induction medium, blue hatched bars represent Sca-1+/CD34+/lin- grown in non-induction medium (α-MEM) and green hatched bars represent Sca-1-/CD34-/lin- cells grown in osteogenic induction medium. (C) Sca-1+/CD34+/lin- cells grown in osteogenic induction medium two weeks post-sort, stained for endogenous alkaline phosphatase. (D) Quantification of alkaline phosphatase staining in cultures by absorbance at 420nm. (E) Sca-1+/CD34+/lin- cells grown in adipogenic medium, two weeks post-sort, stained with Oil Red O. Lipid droplets are stained red. (F) Quantification of Oil Red O stain by absorbance at 519nm. (G) Sca-1+/CD34+/lin- cells grown in chondrogenic induction medium formed pellets two weeks post sorting whereas Sca-1+/CD34+/lin- cells grown in α-MEM and Sca-1-/CD34-/lin- cells grown in osteogenic induction medium did not form pellets (H, J). (I) Cross section of chondrogenic pellet such as in (G) stained with toluidine blue. Asterisks in (B), (D) and (F) represent significance values of P < 0.05 *, P < 0.01 ** and P < 0.001 *** after Student’s T-Tests. Scale bars are as shown.
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pone.0141437.g003: In vitro differentiation of mouse bladder mesenchymal stem cells.(A) Sca-1+/CD34+/lin- cells grown in osteogenic medium, two weeks post-sort, stained with alizarin red for calcium deposition. (B) Quantification of alizarin red by absorbance at 556nm. Red hatched bar represents Sca-1+/CD34+/lin- cells grown in osteogenic induction medium, blue hatched bars represent Sca-1+/CD34+/lin- grown in non-induction medium (α-MEM) and green hatched bars represent Sca-1-/CD34-/lin- cells grown in osteogenic induction medium. (C) Sca-1+/CD34+/lin- cells grown in osteogenic induction medium two weeks post-sort, stained for endogenous alkaline phosphatase. (D) Quantification of alkaline phosphatase staining in cultures by absorbance at 420nm. (E) Sca-1+/CD34+/lin- cells grown in adipogenic medium, two weeks post-sort, stained with Oil Red O. Lipid droplets are stained red. (F) Quantification of Oil Red O stain by absorbance at 519nm. (G) Sca-1+/CD34+/lin- cells grown in chondrogenic induction medium formed pellets two weeks post sorting whereas Sca-1+/CD34+/lin- cells grown in α-MEM and Sca-1-/CD34-/lin- cells grown in osteogenic induction medium did not form pellets (H, J). (I) Cross section of chondrogenic pellet such as in (G) stained with toluidine blue. Asterisks in (B), (D) and (F) represent significance values of P < 0.05 *, P < 0.01 ** and P < 0.001 *** after Student’s T-Tests. Scale bars are as shown.

Mentions: While the exact sorting profile of MSCs is still a topic debate, a hallmark of MSCs is their ability to form colonies and adhere to tissue culture plastic [24]. Because Sca-1+/CD34+/lin- bladder cells both adhered to tissue culture plastic and formed colonies, we next investigated their ability differentiate into various mesenchymal lineages (Fig 3). Here we found that when bladder derived Sca-1+/CD34+/lin- cells were placed in induction media, they formed bone (Fig 3A, 3B, 3C and 3D), adipose (Fig 3E and 3F) and cartilage (Fig 3G and 3I) cells. Brightfield images (S2 Fig; Fig 3H and 3J) coupled with staining (Fig 3B, 3D and 3F) suggest that Sca-1+/CD34+lin- cells cultured in α-MEM alone or Sca-1-/CD34-/lin- cultured in induction medium did not differentiate into these cells types. Thus, EdU incorporation into cells that continued to express Sca-1 and differentiation into bone, cartilage and fat in specialized medium supported the idea that Sca-1+/CD34+/lin- bladder cells could represent a resident MSC population.


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)

In vitro differentiation of mouse bladder mesenchymal stem cells.(A) Sca-1+/CD34+/lin- cells grown in osteogenic medium, two weeks post-sort, stained with alizarin red for calcium deposition. (B) Quantification of alizarin red by absorbance at 556nm. Red hatched bar represents Sca-1+/CD34+/lin- cells grown in osteogenic induction medium, blue hatched bars represent Sca-1+/CD34+/lin- grown in non-induction medium (α-MEM) and green hatched bars represent Sca-1-/CD34-/lin- cells grown in osteogenic induction medium. (C) Sca-1+/CD34+/lin- cells grown in osteogenic induction medium two weeks post-sort, stained for endogenous alkaline phosphatase. (D) Quantification of alkaline phosphatase staining in cultures by absorbance at 420nm. (E) Sca-1+/CD34+/lin- cells grown in adipogenic medium, two weeks post-sort, stained with Oil Red O. Lipid droplets are stained red. (F) Quantification of Oil Red O stain by absorbance at 519nm. (G) Sca-1+/CD34+/lin- cells grown in chondrogenic induction medium formed pellets two weeks post sorting whereas Sca-1+/CD34+/lin- cells grown in α-MEM and Sca-1-/CD34-/lin- cells grown in osteogenic induction medium did not form pellets (H, J). (I) Cross section of chondrogenic pellet such as in (G) stained with toluidine blue. Asterisks in (B), (D) and (F) represent significance values of P < 0.05 *, P < 0.01 ** and P < 0.001 *** after Student’s T-Tests. Scale bars are as shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141437.g003: In vitro differentiation of mouse bladder mesenchymal stem cells.(A) Sca-1+/CD34+/lin- cells grown in osteogenic medium, two weeks post-sort, stained with alizarin red for calcium deposition. (B) Quantification of alizarin red by absorbance at 556nm. Red hatched bar represents Sca-1+/CD34+/lin- cells grown in osteogenic induction medium, blue hatched bars represent Sca-1+/CD34+/lin- grown in non-induction medium (α-MEM) and green hatched bars represent Sca-1-/CD34-/lin- cells grown in osteogenic induction medium. (C) Sca-1+/CD34+/lin- cells grown in osteogenic induction medium two weeks post-sort, stained for endogenous alkaline phosphatase. (D) Quantification of alkaline phosphatase staining in cultures by absorbance at 420nm. (E) Sca-1+/CD34+/lin- cells grown in adipogenic medium, two weeks post-sort, stained with Oil Red O. Lipid droplets are stained red. (F) Quantification of Oil Red O stain by absorbance at 519nm. (G) Sca-1+/CD34+/lin- cells grown in chondrogenic induction medium formed pellets two weeks post sorting whereas Sca-1+/CD34+/lin- cells grown in α-MEM and Sca-1-/CD34-/lin- cells grown in osteogenic induction medium did not form pellets (H, J). (I) Cross section of chondrogenic pellet such as in (G) stained with toluidine blue. Asterisks in (B), (D) and (F) represent significance values of P < 0.05 *, P < 0.01 ** and P < 0.001 *** after Student’s T-Tests. Scale bars are as shown.
Mentions: While the exact sorting profile of MSCs is still a topic debate, a hallmark of MSCs is their ability to form colonies and adhere to tissue culture plastic [24]. Because Sca-1+/CD34+/lin- bladder cells both adhered to tissue culture plastic and formed colonies, we next investigated their ability differentiate into various mesenchymal lineages (Fig 3). Here we found that when bladder derived Sca-1+/CD34+/lin- cells were placed in induction media, they formed bone (Fig 3A, 3B, 3C and 3D), adipose (Fig 3E and 3F) and cartilage (Fig 3G and 3I) cells. Brightfield images (S2 Fig; Fig 3H and 3J) coupled with staining (Fig 3B, 3D and 3F) suggest that Sca-1+/CD34+lin- cells cultured in α-MEM alone or Sca-1-/CD34-/lin- cultured in induction medium did not differentiate into these cells types. Thus, EdU incorporation into cells that continued to express Sca-1 and differentiation into bone, cartilage and fat in specialized medium supported the idea that Sca-1+/CD34+/lin- bladder cells could represent a resident MSC population.

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