Histological analysis of transplanted engineered bladders.

<p>A) Engineered bladders from BAM alone and MSCs-seeded BAMs were retrieved at 1 (not shown) and 6 months (shown) and compared to normal bladders. H&E staining reveals that all bladders possess a tri-layered organization including urothelium, lamina propria and smooth muscle. Masson's trichrome staining shows that unseeded BAMs had more fibrosis (**) and myofibroblastic proliferation (*) compared to normal controls and MSCs-seeded BAMs. IHC for pancytokeratins AE1/3 reveals multilayered urothelium in both unseeded and MSCs-seeded BAMs. IHC for α-SMA shows that smooth muscle fibers in MSCs-seeded BAMs were thicker, more organized and more fascicular than in unseeded BAMs. Scale bars  = 150 µm. B) Histomorphometry of SMA+ area over total tissue volume confirms higher smooth muscle regeneration in MSC-seeded BAM group over BAM alone group at six months post-transplant. C) Urothelium layer thickness was comparable in all groups at six months post-transplant.</p

Performance of engineered bladders <i>in vivo</i>.

<p>A) Gross appearance of MSCs-seeded BAMs immediately after transplantation (0 month) and before retrieval (1 month and 6 months). The anastomosis between native (*) and engineered (**) bladder is indicated by a dashed line. Note that the anastomosis is inconspicuous at 1 and 6 months and that the graft appears normal and well vascularized. B) Representative CMG graphs of animals from normal control group, partial cystectomy (PC) group and MSCs-seeded BAM group. Arrowheads indicate micturition. C) Urodynamic data from all animal tested was plotted and compared using Kruskal-Wallis tests. MSCs-seeded BAMs performed better than unseeded BAMs at 6 months. At 6 months, MSCs-seeded BAMs had restored full bladder capacity when compared to normal controls. NS: not significant. D) Bladder compliance of all animals at 6 months shows that MSC-seeded BAM group outperforms BAM alone group.</p

In vitro characterization of rat MSCs alone and on BAM.

<p>A) Rat mesenchymal stem cells (MSCs) isolated from bone marrow flushes by plastic adherence. These cells where shown to possess typical MSC plasticity <i>in vitro</i>, including the capacity to differentiate into Nile Red/PPARγ positive adipocytes, collagen 1/osteocalcin (OC)/Runx2 positive osteoblasts, and collagen 2/Sox-9/FGFR3 positive chondrocytes. Cells at passage three were used. Scale bars  = 100 µm for all panels. B) Rat MSCs at passage three also possessed the capacity to differentiate into smooth muscle cells in vitro when stimulated with TGF-β, as shown by their upregulation of smooth muscle cell markers calponin and α-SMA. C) Flow cytometry performed on passage four rat MSCs confirmed the cells used in subsequent experiments possessed a typical MSC immunophenotype including expression of SMC markers CD44, CD73 and CD105 and were devoid of endothelial, hematopoietic, and monocytic cells (tested using CD31, CD45 and MAC-1, respectively). D) Bladder tissue was decellularized and seeded with MSCs. After seven days in culture, tissues were formalin-fixed and paraffin-embedded for histological analysis (H&E shown). MSCs were found to adhere to and colonize bladder tissue efficiently, as shown by their broad distribution even deep within tissue. BC: bladder cavity. Scale bar  = 250 µm. E) Rat MSCs found in the middle circular (MC) layer of smooth muscle matrix where found to adopt a smooth muscle cell phenotype, including the expression of the smooth muscle cell-specific protein calponin. Scale bar  = 50 µm. F) Rat MSCs adopt a phenotype specific to their localization within acellular bladder matrix. Cells found within the MC layer of smooth muscle express α-SMA whereas cell attached to the mucosal surface or submucosa remain undifferentiated (not shown). Scale bar  = 100 µm. G) Rat MSCs seeded on BAMs for seven days remain alive and proliferative, as suggested by their expression of the proliferation marker Ki67. Scale bar  = 100 µm.</p

Senescence of multipotent and committed stromal lineages following exposure to IR.

<p>(<b>A</b>) Murine bone marrow-derived multipotent stromal cells (MSC), osteoblasts (OB–SC) and pre-adiopocytes (3T3-L1) were exposed (IR) or not (CTRL) to 10 Gy IR and 7 days later stained for the expression of the senescence-associated β-galactosidase (SAβ-gal). (<b>B</b>) Quantification of the proportion of SAβ-gal positive cells in each population. (<b>C</b>) Sustained activation of the DNA damage response in stromal populations was measured by staining for the presence of 53BP1 DNA damage foci (in red) one week post exposure to IR. Nuclei were counterstained with DAPI. (<b>D</b>) The proliferation capacity of MSC, OB–SC and 3T3-L1 cell population was determined using a CFU assay one week post-exposure or not to IR. Mean ± standard error of at least three individual experiments is shown. p values were obtained by performing a Student’s t-test.</p

Derivation and characterization of BAMs from multiple species.

<p>A) Swine, rabbit and rat bladders were decellularized as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111966#s2" target="_blank">methods</a> section. Detergent treatment alone was not sufficient to remove all cellular debris, in particular nuclei, indicated with black arrows (ii). DNase type I was used to remove residual DNA, followed by extensive washes in PBS (iii). Paraffin sections were stained with hematoxylin-eosin (H&E). In all species tested including thick porcine bladders, this protocol resulted in complete removal of cellular components with no obvious damage to extracellular matrix architecture. Scale bars  = 500 µm (swine), 100 µm (rat). B) Confocal microscopy analysis of decellularized rat bladder. In the smooth muscle region of the bladder, the extracellular matrix protein collagen 1 could be detected as long fibrillar proteins that were preserved after decellularizarion. The smooth muscle cell-specific proteins calponin and α-smooth muscle actin (α-SMA, middle and right panels, respectively) were highly expressed in normal bladder tissue but only residual staining could be observed after treatment. DAPI counterstain (white arrows) was also used to confirm complete removal of DNA after treatment. The extracellular matrix protein collagen 4 was observed surrounding and between CD31+ blood vessels in normal bladders and was preserved in BAM. The basement membrane laminin was mainly observed around CD31+ blood vessels in normal bladder but also faintly between them. Although CD31+ endothelial cells were removed in BAM, laminin was preserved sourronding decellularized blood vessels. Asterisks indicate bladder cavity.</p

Abrogation of osteogenic differentiation potential following irradiation is limited to stromal progenitor cells.

<p>(<b>A</b>) MSC and osteoblasts (OB–SC) were exposed (IR) or not (CTRL) to 10 Gy IR and one week later placed in osteogenic differentiation media for 14 to 21 days. Representative photographs showing mineralization nodules accumulation stained with Alizarin Red S is shown for each population. Scale bar: 2mm. Phase contrast photograph showing the presence of senescent MSC in absence of mineralization is also shown. (<b>B</b>) Quantification of mineralization was determined by the extraction of Alizarin Red S and detection by spectrophotometry. (<b>C</b> and <b>D</b>) Expression of Runx2 and Osx was determined by quantitative real-time PCR using RNA extracted from control and IR-induced senescent MSC and OB–SC populations cultured or not in osteogenic differentiation media. Mean ± standard error; *: <i>p</i> value < 0.05.</p

IR-induced senescent MSC failed to generate bone in vivo.

<p>(<b>A</b>) Schematic of the experiment. Control or IR-induced senescent MSC were mixed with HA/TCP particles along with collagen and injected subcutaneously to the flank of mice. 10 weeks post injection, implants were retrieved from the animals, embedded in plastic, sectioned and stained with Goldner’s trichrome to detect bone formation. (B) Representative images from n= 6 implants per group showing mineralization (Goldner’s trichrome in green) from control or IR-induced senescent MSC. Implants were counterstained with hematoxylin eosin. Scale bar: 300µm.</p

Loss of osteogenic but not adipogenic potential in senescent MSC is p53 dependent.

<p>(<b>A</b>) MSC derived from p53 knockout mice (MSC-p53KO) were exposed (IR) or not (CTRL) to 10 Gy IR and one week later stained for the expression of SAβ-gal activity. (<b>B</b>) The proliferation capacity of MSC-p53KO was determined using a CFU assay one week post-exposure or not to IR. (<b>C</b>) One week post exposure or not to IR, MSC-p53KO were placed in adipogenic differentiation media for 7 to 14 days. Representative photographs showing lipid accumulation stained with Oil Red O is shown. Scale bar: 200µm. (<b>D</b>) Quantification of lipid accumulation has determined by the extraction of Oil Red O staining and detection by spectrophotometry. (<b>E</b>) Expression of PPARγ was determined by quantitative real-time PCR using RNA extracted from control and IR-induced senescent MSC-p53KO cultured in adipogenic differentiation media. (<b>F</b>) One week post exposure or not to IR, MSC-p53KO were placed in osteogenic differentiation media for 14 to 21 days. Representative photographs showing mineralization nodules accumulation stained with Alizarin Red S is shown. (<b>G</b>) Quantification of mineralization was determined by the extraction of Alizarin Red S staining and detection by spectrophotometry. (<b>H</b>) Expression of Runx2 and Osx was determined by quantitative real-time PCR using RNA extracted from control and IR-induced senescent MSC-p53KO populations placed in osteogenic differentiation media. Mean ± standard error of at least 3 individual experiments is shown; *: <i>p</i> value < 0.05.</p