Multipotential differentiation of human urine-derived stem cells: potential for therapeutic applications in urology.

We sought to biologically characterize and identify a subpopulation of urine-derived stem cells (USCs) with the capacity for multipotent differentiation. We demonstrated that single USCs can expand to a large population with 60-70 population doublings. Nine of 15 individual USC clones expressed detectable levels of telomerase and have long telomeres. These cells expressed pericyte and mesenchymal stem cell markers. Upon induction with appropriate media in vitro, USCs differentiated into bladder-associated cell types, including functional urothelial and smooth muscle cell lineages. When the differentiated USCs were seeded onto a scaffold and subcutaneously implanted into nude mice, multilayered tissue-like structures formed consisting of urothelium and smooth muscle. Additionally, USCs were able to differentiate into endothelial, osteogenic, chondrogenic, adipogenic, skeletal myogenic, and neurogenic lineages but did not form teratomas during the 1-month study despite telomerase activity. USCs may be useful in cell-based therapies and tissue engineering applications, including urogenital reconstruction.

Implantation of autologous urine derived stem cells expressing vascular endothelial growth factor for potential use in genitourinary reconstruction.

PURPOSE: We evaluated the effects of vascular endothelial growth factor overexpression on urine derived stem cell survival and myogenic differentiation to determine whether these cells could be used as a novel cell source for genitourinary reconstruction. MATERIALS AND METHODS: Urine derived stem cells were isolated from 31 urine samples of 6 healthy individuals 3 to 27 years old. Urine derived stem cells were infected with an adenoviral vector containing the mouse VEGF gene. These cells were then mixed with human umbilical vein endothelial cells (total 5×10(6)) in a collagen-I gel. These cell containing gels were subcutaneously implanted along with 6 other controls into 18 athymic mice. The grafts were assessed up to 28 days after injection for gross appearance and immunocytochemistry. RESULTS: Vascular endothelial growth factor levels in the media from infected urine derived stem cell cultures reached a peak value on day 10 after infection. Grafts composed of urine derived stem cell/adenoviral vector containing the mouse VEGF gene and human umbilical vein endothelial cells were larger and better vascularized compared to uninfected urine derived stem cell control grafts. Additionally more implanted cells expressed human nuclear markers in the vascular endothelial growth factor expressing grafts. Vascular endothelial growth factor expressing grafts also contained more cells expressing the endothelial markers CD-31 and von Willebrand factor, and smooth muscle markers (α-smooth muscle actin, desmin and myosin). Also, more nerve fibers were present in urine derived stem cell/adenoviral vector containing mouse VEGF gene plus human umbilical vein endothelial cell grafts than in controls. CONCLUSIONS: Vascular endothelial growth factor overexpression combined with human umbilical vein endothelial cells enhanced in vivo survival and myogenic differentiation of urine derived stem cells. Neovascularization and nerve regeneration were also enhanced within the implanted grafts.

Skeletal myogenic differentiation of human urine-derived cells as a potential source for skeletal muscle regeneration.

Stem cells are regarded as possible cell therapy candidates for skeletal muscle regeneration. However, invasive harvesting of those cells can cause potential harvest-site morbidity. The goal of this study was to assess whether human urine-derived stem cells (USCs), obtained through non-invasive procedures, can differentiate into skeletal muscle linage cells (Sk-MCs) and potentially be used for skeletal muscle regeneration. In this study, USCs were harvested from six healthy individuals aged 25-55. Expression profiles of cell-surface markers were assessed by flow cytometry. To optimize the myogenic differentiation medium, we selected two from four different types of myogenic differentiation media to induce the USCs. Differentiated USCs were identified with myogenic markers by gene and protein expression. USCs were implanted into the tibialis anterior muscles of nude mice for 1 month. The results showed that USCs displayed surface markers with positive staining for CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133, CD146, SSEA-4 and STRO-1, and negative staining for CD14, CD31, CD34 and CD45. After myogenic differentiation, a change in morphology was observed from 'rice-grain'-like cells to spindle-shaped cells. The USCs expressed specific Sk-MC transcripts and protein markers (myf5, myoD, myosin, and desmin) after being induced with different myogenic culture media. Implanted cells expressed Sk-MC markers stably in vivo. Our findings suggest that USCs are able to differentiate into the Sk-MC lineage in vitro and after being implanted in vivo. Thus, they might be a potential source for cell injection therapy in the use of skeletal muscle regeneration. Copyright © 2014 John Wiley & Sons, Ltd.

Resensitization of breast cancer cells to anoikis by tropomyosin-1: role of Rho kinase-dependent cytoskeleton and adhesion.

Two most common properties of malignant cells are the presence of aberrant actin cytoskeleton and resistance to anoikis. Suppression of several key cytoskeletal proteins, including tropomyosin-1 (TM1), during neoplastic transformation is hypothesized to contribute to the altered cytoskeleton and neoplastic phenotype. Using TM1 as a paradigm, we have shown that cytoskeletal proteins induce anoikis in breast cancer (MCF-7 and MDA MB 231) cells. Here, we have tested the hypothesis that TM1-mediated cytoskeletal changes regulate integrin activity and the sensitivity to anoikis. TM1 expression in MDA MB 231 cells promotes the assembly of stress fibers, induces rapid anoikis via caspase-dependent pathways involving the release of cytochrome c. Further, TM1 inhibits binding of MDA MB 231 cells to collagen I, but promotes adhesion to laminin. Inhibition of Rho kinase disrupts TM1-mediated cytoskeletal reorganization and adhesion to the extracellular matrix components, whereas the parental cells attach to collagen I, spread and form extensive actin meshwork in the presence of Rho kinase inhibitor, underscoring the differences in parental and TM1-transduced breast cancer cells. Further, treatment with the cytoskeletal disrupting drugs rescues the cells from TM1-induced anoikis. These new findings demonstrate that the aberrant cytoskeleton contributes to neoplastic transformation by conferring resistance to anoikis. Restoration of stress fiber network through enhanced expression of key cytoskeletal proteins may modulate the activity of focal adhesions and sensitize the neoplastic cells to anoikis.

Loss of expression of tropomyosin-1, a novel class II tumor suppressor that induces anoikis, in primary breast tumors.

Suppression of tropomyosins (TMs), a family of actin-binding, microfilament-associated proteins, is a prominent feature of many transformed cells. Yet it is unclear whether downregulation of TMs occur in human tumors. We have investigated the expression of tropomyosin-1 (TM1) in human breast carcinoma tissues by in situ hybridization and immunofluorescence. TM1 mRNA and protein are readily detectable in normal mammary tissue. In contrast, TM1 expression is abolished in the primary human breast tumors. Expression of other TM isoforms, however, is variable among the tumors. The consistent and profound downregulation of TM1 suggests that TM1 may be a novel and useful biomarker of mammary neoplasms. These data also support the hypothesis that suppression of TM1 expression during the malignant conversion of mammary epithelium as a contributing factor of breast cancer. In support of this hypothesis, we show that the ability to suppress malignant growth properties of breast cancer cells is specific to TM1 isoform. Investigations into the mechanisms of TM1-induced tumor suppression reveal that TM1 induces anoikis (detachment induced apoptosis) in breast cancer cells. Downregulation of TM1 in breast tumors may destabilize microfilament architecture and confer resistance to anoikis, which facilitates survival of neoplastic cells outside the normal microenvironment and promote malignant growth.

Amino terminal, but not the carboxy terminal, sequences of tropomyosin-1 are essential for the induction of stress fiber assembly in neoplastic cells.

The presence of aberrant cytoskeleton, arising from the downregulation of key cytoskeletal proteins such as tropomyosins (TMs), is a prominent feature of many malignant cells and is suggested to promote neoplastic growth. While our previous work demonstrated that tropomyosin-1 (TM1) promotes stress fiber assembly and suppresses malignant growth, the molecular basis of the anti-oncogenic effects of TM1 has not been determined. By employing chimeric TMs, here we demonstrate that the amino terminal portion of TM1, but not the carboxy terminal portion which contains the alternatively spliced exon-coded sequences, is essential for stress fiber assembly and suppression of malignant growth. These studies also indicate that the amino and carboxy termini of TM1 coordinately function to regulate microfilament organization during cytokinesis.

Tropomyosin-1, a novel suppressor of cellular transformation is downregulated by promoter methylation in cancer cells.

Tropomyosins (TMs) are a family of microfilament binding proteins, which are suppressed in the transformed cells. We have investigated the mechanism of suppression of TMs, in particular that of tropomyosin-1 (TM1), in breast cancer cells. Inhibition of DNA methyl transferase with 5-aza-2'-deoxycytidine (AZA) alone did not induce TM1 expression. However, combined treatment of trichostatin A (TSA) and AZA resulted in readily detectable expression of TM1, but not that of other TM isoforms. Upregulation of TM1 expression paralleled with the reemergence of TM1 containing microfilaments, and in abolition of anchorage-independent growth. The synergistic action of AZA and TSA in reactivation of TM1 gene was also evident in ras-transformed fibroblasts. These data, for the first time, show that hypermethylation of TM1 gene and chromatin remodeling are the predominant mechanisms by which TM1 expression is downregulated in breast cancer cells.

Isolation and myogenic differentiation of mesenchymal stem cells for urologic tissue engineering.

Cell-based tissue engineering is one of the most promising areas in biotechnology for restoring tissues and organ function in the urinary tract. Current strategies for bladder tissue engineering require a competent biological scaffold that is seeded in vitro with the patient's own bladder cells. This use of autologous cells avoids graft rejection and the long-term use of immunosuppressive medications usually required after allogeneic transplantation. However, suitable bladder cells from the patient are sometimes limited or unobtainable. When suitable cells are unavailable for seeding due to bladder exstrophy, malignancy, or other reasons, the use of other cell types originating from the patient may be an alternative. A suitable alternative to autologous bladder cells could be mesenchymal stem cells (MSC). MSC reside primarily in the bone marrow, although they exist in other sites as well, including adipose tissue, peripheral and cord blood, liver tissue, and fetal tissues. Bone marrow-derived stromal cell populations contain few MSC (one MSC in 10(4)-5 × 10(7) marrow cells), with the exact number depending on the age of the patient. Despite their limited numbers, MSC possess both the ability to self-renew for extended periods of time and the potential to differentiate into several different specialized cell types under the appropriate conditions. MSC are capable of expansion and tissue-specific differentiation in vitro based on external signals and/or the environment. There are different methodologies for induction and maintenance of a differentiated cell phenotype from MSC. For example, MSC can differentiate into a smooth muscle cell (SMC) phenotype in vitro when exposed to stimuli such as conditioned medium derived from SMC cultures or specific myogenic growth factors (PDGF-BB, HGF, TGF-ß). These differential cells can migrate to a scaffold for differentiation into smooth muscle-like cells in vivo. Furthermore, stem cell-seeded scaffolds that are implanted into the bladders repopulate and reorganize the tissue rapidly, thus reducing fibrosis and restoring appropriate neural functionality.In this chapter, we describe the methods we use for the isolation of human bone marrow mesenchymal stem cells (BMSC), and demonstrate evidence of their myogenic differentiation capacity for potential use in urologic tissue engineering.

Self-renewal and differentiation capacity of urine-derived stem cells after urine preservation for 24 hours.

Despite successful approaches to preserve organs, tissues, and isolated cells, the maintenance of stem cell viability and function in body fluids during storage for cell distribution and transportation remains unexplored. The aim of this study was to characterize urine-derived stem cells (USCs) after optimal preservation of urine specimens for up to 24 hours. A total of 415 urine specimens were collected from 12 healthy men (age range 20-54 years old). About 6 × 10(4) cells shed off from the urinary tract system in 24 hours. At least 100 USC clones were obtained from the stored urine specimens after 24 hours and maintained similar biological features to fresh USCs. The stored USCs had a "rice grain" shape in primary culture, and expressed mesenchymal stem cell surface markers, high telomerase activity, and normal karyotypes. Importantly, the preserved cells retained bipotent differentiation capacity. Differentiated USCs expressed myogenic specific proteins and contractile function when exposed to myogenic differentiation medium, and they expressed urothelial cell-specific markers and barrier function when exposed to urothelial differentiation medium. These data demonstrated that up to 75% of fresh USCs can be safely persevered in urine for 24 hours and that these cells stored in urine retain their original stem cell properties, indicating that preserved USCs could be available for potential use in cell-based therapy or clinical diagnosis.

Characterizing the micro-scale elastic modulus of hydrogels for use in regenerative medicine.

Our objective was to characterize the elasticity of hydrogel formulations intended to mimic physical properties that cells and tissues experience in vivo. Using atomic force microscopy (AFM), we tested a variety of concentrations in a variety of biomaterials, including agarose, alginate, the collagens, fibrin, hyaluronic acid, kerateine, laminin, Matrigel, polyacrylamide, polyethylene glycol diacrylate (PEGDA) and silicone elastomer (polydimethylsiloxane). Manipulations of the concentration of biomaterials were detectable in AFM measurements of elasticity (Young's modulus, E), and E tended to increase with increased concentration. Depending on the biomaterials chosen, and their concentrations, generation of tunable biocompatible hydrogels in the physiologic range is possible.

Tissue specific synthetic ECM hydrogels for 3-D in vitro maintenance of hepatocyte function.

Despite recent advances in biomaterial science, there is yet no culture system that supports long-term culture expansion of human adult hepatocytes, while preserving continued function. Previous studies suggested that acellular liver extracellular matrix (ECM), employed as a substrate, improved proliferation and function of liver cells. Here we investigated whether extracts prepared from acellular liver ECM (liver ECM extract, LEE), or from whole (fresh) liver tissue (liver tissue extract, LTE), could be combined with collagen Type I, hyaluronic acid (HA), or heparin-conjugated HA (HP) hydrogels to enhance survival and functional output of primary human hepatocytes. The liver-specific semi-synthetic ECMs (sECMs) were prepared by incorporating LEE or LTE into the gel matrices. Subsequently, primary human hepatocytes were maintained in sandwich-style hydrogel cultures for 4 weeks. Progressive increase in hepatocyte metabolism was observed in all HA and HP groups. Hepatocytes cultured in HA and HP hydrogels containing LEE or LTE synthesized and secreted steady levels of albumin and urea and sustained cytochrome p450-dependent drug metabolism of ethoxycoumarin. Collectively, these results indicate that customized HA hydrogels with liver-specific ECM components may be an efficient method for expansion human hepatocytes in vitro for cell therapy and drug and toxicology screening purposes.

Human urine-derived stem cells seeded in a modified 3D porous small intestinal submucosa scaffold for urethral tissue engineering.

The goal of this study was to determine whether urothelial cells (UC) and smooth muscle cells (SMC) derived from the differentiation of urine-derived stem cells (USC) could be used to form engineered urethral tissue when seeded on a modified 3-D porous small intestinal submucosa (SIS) scaffold. Cells were obtained from 12 voided urine samples from 4 healthy individuals. USC were isolated, characterized and induced to differentiate into UC and SMC. Fresh SIS derived from pigs was decellularized with 5% peracetic acid (PAA). Differentiated UC and SMC derived from USC were seeded onto SIS scaffolds with highly porous microstructure in a layered co-culture fashion and cultured under dynamic conditions for one week. The seeded cells formed multiple uniform layers on the SIS and penetrated deeper into the porous matrix during dynamic culture. USC that were induced to differentiate also expressed UC markers (Uroplakin-III and AE1/AE3) or SMC markers (α-SM actin, desmin, and myosin) after implantation into athymic mice for one month, and the resulting tissues were similar to those formed when UC and SMC derived from native ureter were used. In conclusion, UC and SMC derived from USC could be maintained on 3-D porous SIS scaffold. The dynamic culture system promoted 3-D cell-matrix ingrowth and development of a multilayer mucosal structure similar to that of native urinary tract tissue. USC may serve as an alternative cell source in cell-based tissue engineering for urethral reconstruction or other urological tissue repair.

Characterization of urine-derived stem cells obtained from upper urinary tract for use in cell-based urological tissue engineering.

BACKGROUND: The goals of this study were to characterize urine-derived stem cells obtained from the upper urinary tract (uUSC), induce these cells to differentiate into urothelial and smooth muscle cells, and determine whether they could serve as a potential stem cell source for bladder tissue engineering. MATERIALS AND METHODS: Urine samples were collected from five patients with normal upper urinary tracts during renal pyeloplasty. Cells were isolated from this urine and extensively expanded in vitro. RESULTS: The mean population doubling of uUSC was 46.5±7.7. The uUSC expressed surface markers associated with mesenchymal stem cells and pericytes. These cells could differentiate into smooth muscle-like cells that expressed smooth muscle-specific gene transcripts and proteins, including α-smooth muscle actin, desmin, and myosin, when exposed to TGF-ß1 and PDGF-BB. In a collagen lattice assay, these myogenic-differentiated uUSC displayed contractile function that was similar to that seen in native smooth muscle cells. Urothelial-differentiated uUSC expressed urothelial-specific genes and proteins such as uroplakin-Ia and -III, cytokeratin (CK)-7, and CK-13. CONCLUSIONS: uUSC possess expansion and differentiation (urothelial and myogenic) capabilities, and can potentially be used as an alternative cell source in bladder tissue engineering for patients needing cystoplasty.

Three-dimensional culture of hepatocytes on porcine liver tissue-derived extracellular matrix.

There is currently no optimal system to expand and maintain the function of human adult hepatocytes in culture. Recent studies have demonstrated that specific tissue-derived extracellular matrix (ECM) can serve as a culture substrate and that cells tend to proliferate and differentiate best on ECM derived from their tissue of origin. The goal of this study was to investigate whether three-dimensional (3D) ECM derived from porcine liver can facilitate the growth and maintenance of physiological functions of liver cells. Optimized decellularization/oxidation procedures removed up to 93% of the cellular components from porcine liver tissue and preserved key molecular components in the ECM, including collagen-I, -III, and -IV, proteoglycans, glycosaminoglycans, fibronectin, elastin, and laminin. When HepG2 cells or human hepatocytes were seeded onto ECM discs, uniform multi-layer constructs of both cell types were formed. Dynamic culture conditions yielded better cellular infiltration into the ECM discs. Human hepatocytes cultured on ECM discs expressed significantly higher levels of albumin over a 21-day culture period compared to cells cultured in traditional polystyrene cultureware or in a collagen gel "sandwich". The culture of hepatocytes on 3D liver-specific ECM resulted in considerably improved cell growth and maintained cell function; therefore, this system could potentially be used in liver tissue regeneration, drug discovery or toxicology studies.

Tissue-engineered conduit using urine-derived stem cells seeded bacterial cellulose polymer in urinary reconstruction and diversion.

The objective of this study was to generate bacterial cellulose (BC) scaffolds seeded with human urine-derived stem cells (USC) to form a tissue-engineered conduit for use in urinary diversion. Microporous BC scaffolds were synthesized and USC were induced to differentiate into urothelial and smooth muscle cells (SMC). Induced USC (10(6) cells/cm(2)) were seeded onto BC under static and 3D dynamic (10 or 40 RPM) conditions and cultured for 2 weeks. The urothelial cells and SMC derived from USC formed multilayers on the BC scaffold surface, and some cells infiltrated into the scaffold. The urothelium derived from USC differentiation expressed urothelial markers (uroplakin Ia and AE1/AE3) and the SMC expressed SMC markers (α-smooth muscle actin and desmin). In addition, USC/BC scaffold constructs were implanted into athymic mice, and the cells were tracked using immunohistochemical staining for human nuclear antigen. In vivo, the cells appeared to differentiate and express urothelial and SMC markers. In conclusion, porous BC scaffolds allow 3 dimensional growth of USC, leading to formation of a multilayered urothelium and cell-matrix infiltration. Thus, cell-seeded BC scaffolds hold promise for use in tissue-engineered urinary conduits for urinary reconstruction.

Immunofluorescence microscopy for imaging of nuclear p63 in human primary keratinocytes: a comparison of antibodies and fixation methods.

The ability of an experimental treatment to induce primitive, undifferentiated stem cells towards an epidermal fate may be tested by comparing the treated stem cells with a positive control, such as primary keratinocytes. In an effort to perfect methods used for this comparison, we tested two commercially available antibodies and three fixation methods to determine which antibody/fixation interaction produced the best immunofluorescent images of the nuclear localization of p63, a canonical marker of epidermal fate, in keratinocytes. Here, we report the methods used, and the experimental outcome.

Differentiation of human bone marrow mesenchymal stem cells into bladder cells: potential for urological tissue engineering.

Bone marrow mesenchymal stem cells (BMSCs) are capable of differentiating into multiple cell types, providing an alternative cell source for cell-based therapy and tissue engineering. Simultaneous differentiation of human BMSCs into smooth muscle cells (SMCs) and urothelium would be beneficial for clinical applications in bladder regeneration for patients with bladder exstrophy or cancer who need cystoplasty. We investigated the ability of human BMSCs to differentiate toward both SMCs and urothelium with cocultured or conditioned media and analyzed growth factors from a coculture system. After being cocultured with urothelium or cultured using urothelium-derived conditioned medium, human BMSCs expressed urothelium-specific genes and proteins: uroplakin-Ia, cytokeratin-7, and cytokeratin-13. When cocultured with SMCs or cultured in SMC-conditioned medium, human BMSCs expressed SMC-specific genes and proteins: desmin and myosin. Several growth factors (hepatocyte growth factor, platelet-derived growth factor-homodimer polypeptide of B chain (BB), transforming growth factor-beta1, and vascular endothelial growth factor) were detected in the SMC cocultured media and in the urothelium cocultured media (epidermal growth factor, platelet-derived growth factor-BB, transforming growth factor-beta1, and vascular endothelial growth factor). BMSC-scaffold constructs significantly improved cell contractility after myogenic differentiation. In vivo-grafted cells displayed significant matrix infiltration and expressed SMC-specific markers in the nanofibrous poly-l-lactic acid scaffolds. In conclusion, smooth muscle- and urothelium-like cells derived from human BMSCs provide an alternative cell source for potential use in bladder tissue engineering.

Myogenic differentiation of human bone marrow mesenchymal stem cells on a 3D nano fibrous scaffold for bladder tissue engineering.

Current strategies for engineering bladder tissues include a bladder biopsy for in vitro cell expansion for use in reconstructive procedures. However, this approach cannot be used in patients with bladder cancer who need a complete bladder replacement. Bone marrow mesenchymal stem cells (BMSC) might be an alternative cell source to better meet this need. We investigated the effects of soluble growth factors, bladder extracellular matrix (ECM), and 3D dynamic culture on cell proliferation and differentiation of human BMSC into smooth muscle cells (SMC). Myogenic growth factors (PDGF-BB and TGF-beta1) alone, or combined either with bladder ECM or dynamic cultures, induced BMSC to express smooth muscle-specific genes and proteins. Either ECM or the dynamic culture alone promoted cell proliferation but did not induce myogenic differentiation of BMSC. A highly porous poly-l-lactic acid (PLLA) scaffold provided a 3D structure for maximizing the cell-matrix penetration, maintained myogenic differentiation of the induced BMSC, and promoted tissue remolding with rich capillary formation in vivo. Our results demonstrate that myogenic-differentiated BMSC seeded on a nano fibrous PLLA scaffold can be potentially used for cell-based tissue engineering for bladder cancer patients requiring cystoplasty.

Optimization of a natural collagen scaffold to aid cell-matrix penetration for urologic tissue engineering.

The goal of this study was to fabricate a 3-dimensional (3-D) porous scaffold derived from bladder submucosa (BSM) and further recellularize the scaffold with human bladder cells for cell-based urethral tissue engineering. Fresh porcine BSM was soaked with peracetic acid (PAA) at different concentrations (0,1,3,5 and 10%) and then treated with Triton X-100 for decellularization. DNA content analysis showed that nuclear material was removed from the BSM scaffold. Treatment with 5% PAA led to high porosity on the surface of the matrix with retention of less cellular material and maintained about 75% of normal tensile strength. In 3-D dynamic culture, cells formed even multiple layers on the surface of matrix. Cells also penetrated deeper into the lamina propria of the matrix compared to untreated matrix. Immunocytochemical staining indicated that the grafted bladder cells expressed urothelial- and smooth muscle-specific markers both, in vitro and in vivo. This study demonstrates that decellularized/oxidized BSM possesses 3-D porosity for cell infiltration into the matrix. Further, cells seeded on decellularized/oxidized BSM and grown in dynamic culture, significantly promoted cell-matrix penetration in vitro and promoted cell growth in vivo. Scaffolds with such characteristics have potential applications in cell-based urological tissue engineering.

Tissue-specific extracellular matrix coatings for the promotion of cell proliferation and maintenance of cell phenotype.

Recent studies have shown that extracellular matrix (ECM) substitutes can have a dramatic impact on cell growth, differentiation and function. However, these ECMs are often applied generically and have yet to be developed for specific cell types. In this study, we developed tissue-specific ECM-based coating substrates for skin, skeletal muscle and liver cell cultures. Cellular components were removed from adult skin, skeletal muscle, and liver tissues, and the resulting acellular matrices were homogenized and dissolved. The ECM solutions were used to coat culture dishes. Tissue matched and non-tissue matched cell types were grown on these coatings to assess adhesion, proliferation, maintenance of phenotype and cell function at several time points. Each cell type showed better proliferation and differentiation in cultures containing ECM from their tissue of origin. Although subtle compositional differences in the three ECM types were not investigated in this study, these results suggest that tissue-specific ECMs provide a culture microenvironment that is similar to the in vivo environment when used as coating substrates, and this new culture technique has the potential for use in drug development and the development of cell-based therapies.