Adipose-derived stem cells (ADSCs) and muscle precursor cells (MPCs) for the treatment of bladder voiding dysfunction

Purpose: Bladder outflow obstruction (BOO) is common in the elderly and can result in bladder voiding dysfunction (BVD) due to severe bladder muscle damage. The goal of this research was to evaluate the use of adult stem cells for the treatment of BVD due to decreased muscle contractility in a rat model. Materials and methods: Adipose-derived stem cells (ADSCs) and muscle precursor cells (MPCs) were harvested from male Lewis rats and expanded in culture. BOO was induced by tying a suture around the urethra. Six weeks after obstruction, the development of BVD was confirmed by cystometric analysis in conscious rats, histology and molecular investigations. Injection of ADSCs or MPCs into the bladder wall and synchronous deligation was performed 6weeks after the obstruction. After stem-cell treatment, morphological and functional changes were assessed. Age-matched rats and animals without cellular therapy but deligation-only served as controls. Results: Voiding pressures decreased progressively 6weeks after obstruction with increased bladder capacities. Structural changes of the detrusor muscle occurred during the time of obstruction with an increased connective tissue-to-smooth muscle ratio and decreased SMA/smoothelin expression. After stem-cell injection, improved voiding pressures and voiding volumes were observed together with recovered tissue architecture. RT-PCR and Western blotting showed an up-regulation of important contractile proteins. Conclusions: We established a reliable model for BVD and demonstrated that ADSCs and MPCs can prevent pathophysiological remodelling and provide regenerated bladder tissue and function

Muscle precursor cells for the restoration of irreversibly damaged sphincter function

Multiple modalities, including injectable bulking agents and surgery, have been used to treat stress urinary incontinence. However, none of these methods is able to fully restore normal striated sphincter muscle function. In this study, we explored the possibility of achieving functional recovery of the urinary sphincter muscle using autologous muscle precursor cells (MPCs) as an injectable, cell-based therapy. A canine model of striated urinary sphincter insufficiency was created by microsurgically removing part of the sphincter muscle in 24 dogs. Autologous MPCs were obtained, expanded in culture and injected into the damaged sphincter muscles of 12 animals. The animals were followed for up to 6 months after injection, and urodynamic studies, functional organ bath studies, ultrastructural and histological examinations were performed. Animals receiving MPC injections demonstrated sphincter pressures of approximately 80% of normal values, while the pressures in the control animals without cells dropped and remained at 20% of normal values. Histological analysis indicated that the implanted cells survived and formed tissue, including new innervated muscle fibers, within the injected region of the sphincter. These results indicate that autologous muscle precursor cells may be able to restore otherwise irreversibly damaged urinary sphincter function clinically

Ultrastructural analysis of renal proximal tubular cells using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM).

<p>SEM analysis showing long microvilli on the apical surface membrane of a Proximal tubular cells derived from a NK kidney at passage 3 (A) and passage 6 (C) and the same cell type from a CKD kidney at passage 3 (B) and passage 6 (D); Upper panel shows the cellular morphology magnified x630. TEM analysis showing the integrity of tight junction (arrow) in proximal tubular cells isolated from primary renal NK kidneys (E) and CKD kidney cells (F) at passage 3 (P3) magnified x4780; TEM micrograph showing the ultrastructure of nucleus “N” and other intracellular components in proximal tubular cells of NK (G) and CKD (H) kidneys are similar morphology, magnified x11000.</p

Potential Use of Autologous Renal Cells from Diseased Kidneys for the Treatment of Renal Failure - Fig 6

<p>Photomicrograph of proximal tubular cells (PTC) purified from primary cell cultures that were originally derived from NK kidneys (A) and CKD kidneys (B) at passage 1 (P1) original magnification x10. Consolidated growth curve of proximal tubular cells isolated from NK and CKD human renal cells (C). Proximal tubular cells from different age donors were counted after achieving confluency, had the same behavior in culture.</p

Identification of Podocytes among the primary renal cells from NK and CKD using antibody-based staining of cell surface markers.

<p>(A-B) Podocytes and endothelial cells identified by Podocalyxin (PDX) staining; (C-D) Podocytes stained using Wilms’ tumor (WT-1) antibody; (E-F) Staining of Podocytes using another cell-specific marker Nephrin; Quantitation of Podocytes among the primary renal cells isolated from NK and CKD kidneys at P3. Note that use of WT-1 and Nephrin antibodies resulted in slightly different levels of Podocytes detection amount the renal cell population (G). However, the relative amounts of Podocytes in both NK and CKD kidney derived cells were similar.</p

Characterization of specific renal cells among the total renal cells isolated from NK and CKD kidneys.

<p>Cell-type specific antibodies and FACS was used to isolate proximal tubular cells (A-B), distal tubular cells (D-E) and podocytes (G-H) in different passages (P3 and P9) of cultured renal cells from NK and CKD kidneys. FACS-based quantification percentage of proximal tubular cells in passage 3 and 9 cells (C), percentage of distal tubular cells (F), percentage of podocytes (I). The result highlights that there is no significant difference between the groups.</p

Oxidative Stress in NK and CKD kidney derived tissues.

<p>Immunohistochemical staining to detect Superoxide dismutase 1 (SOD1) expression in human renal tissues from NK (A) and CKD (B) kidneys. Quantitation of oxidative stress in primary renal cells derived from NK and CKD kidneys using a Glutathione (GSH) assay that utilizes a fluorescent dye Monochlorobimane (MCB), (C). GSH was assayed in cells of passage 3 (P3) to (P9) and 12. The Glutathione levels in renal cells derived from both NK and CKD kidneys were almost similar during the entire cell culture</p

Summary of donor information and disease status.

<p>Summary of donor information and disease status.</p

Quantitative uptake analysis of primary proximal tubular cells (PTC) derived from NK and CKD kidneys confirms specific uptake of Na⁺ by the cells with no significant difference.

<p>Ouabain treatment increases sodium uptake by inhibiting Na/K ATPase. FACS analysis of Intracellular Na⁺ uptake using the cell permeant Sodium Green Tetra-acetate in (PTC) cells from NK and CKD kidneys (B-C) were similar.</p

Characterization of isolated primary renal cells from NK and CKD kidneys using cell-specific markers.

<p>Florescent antibody staining was carried out on passage 3 (P3) cells. Staining with proximal tubular marker Aquaporin-1 (A-B); Quantitation of proximal tubular cells (Aquaporin-1) among the total isolated primary renal cells at different passages from P3 to P12; (C) Distal tubular marker E-cadherin1 staining of primary renal cells from NK and CKD kidneys. N = 3; (D-E) Quantitation of distal tubular cell (E-cadherin) among the total isolated primary renal cells at different passages from P3 to P12 (F). The overall amounts (percentage) of proximal tubular cells and distal tubular cell were similar in the renal cell population derived from NK and CKD kidneys. Original magnification x20.</p