Type IV Collagen Induces Podocytic Features in Bone Marrow Stromal Stem Cells In Vitro Julie Perry,* Stephanie Tam,* Keqin Zheng,* Yoshikazu Sado, † Howard Dobson, ‡ Barbara Jefferson, § Robert Jacobs, § Paul S. Thorner*  *Division of Pathology, Hospital for Sick Children, Toronto, Ontario, Canada; † Division of Immunology, Shigei Medical Research Institute, Okayama, Japan; Departments of ‡ Clinical Studies and § Pathobiology, University of Guelph, Guelph, Ontario, Canada; and  Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Bone marrow– derived stromal stem cells (BMSC) can differentiate along a variety of mesenchymal lines, including mesangial cells. For determining whether BMSC can be induced to differentiate along podocytic lines in vitro, canine BMSC were cultured on plastic, type I collagen, and NC1 hexamers of type IV collagen from normal and Alport canine glomerular basement membrane. Results were compared with a mouse podocyte cell line. In the case of the podocyte line, differentiation occurred on all three matrices as indicated by the expression of synaptopodin and CD2-associated protein (CD2AP) and organization of myosin heavy chain IIA into a linear pattern. BMSC proliferated equally well on all matrices, but cells that were grown on type IV collagen NC1 hexamers became larger and stellate. Evidence for podocytic differentiation occurred on all three collagen matrices as indicated by the redistribution of myosin IIA to a linear pattern and expression of synaptopodin, CD2AP, and -actinin. A punctate distribution of CD2AP was seen only in cells that were grown on normal and Alport glomerular basement membrane NC1 hexamers. Differentiated podocytes expressed the 1, 2, and 5 chains of type IV collagen but at higher levels in cells that were grown on NC1 hexamers. Similar results were obtained in BMSC for the 1 and 2 chains only. The 3, 4, and 6 chains were never detected in the podocyte line or BMSC. These results indicate that BMSC undergo a degree of podocytic differentiation in vitro and greater when grown on type IV collagen NC1 hexamers than type I collagen. Alport and normal NC1 hexamers seem equally permissive to BMSC growth and differentiation, suggesting that these processes are not influenced specifically by the 3/4/5 network. BMSC may be useful in the development of stem cell– based reconstitution of glomeruli that are damaged by disease and for gene therapy of genetic glomerular diseases such as Alport syndrome. J Am Soc Nephrol 17: 66 –76, 2006. doi: 10.1681/ASN.2005060586 B one marrow– derived stromal stem cells (BMSC) can exhibit considerable phenotypic plasticity, including mesenchymal differentiation (osteocyte, adipocyte, and chondrocyte) and “unorthodox” differentiation (hepatocyte, muscle, and neural) (1–3). BMSC have been shown to take up residence in glomeruli in vivo and differentiate into mesangial cells and even podocytes (4 – 8). Such cells hold promise for reconstitution of diseased glomeruli and could conceivably be used to deliver a transgene to the glomerulus for gene therapy. It is known that specific microenvironments can induce BMSC to differentiate along particular mesenchymal lines (2). The available data on glomeruli are largely based on experi- ments that were designed to reconstitute bone marrow with the detection of “stray” cells that repopulated glomeruli. Because these BMSC are physically distant from their bone marrow origin, it is reasonable to postulate that the microenvironment of the kidney promoted the differentiation of such cells into glomerular cells. The conditions that might favor glomerular cell differentiation from these multipotential cells are poorly understood. PDGF- induces mesangial cell differentiation in cultured stromal stem cells that are grown on type IV collagen (9), but experimental data on differentiation of stromal stem cells into podocytes are lacking. Many of the proteins that are expressed in mature podocytes are specific or nearly specific to that one cell type and offer an approach to assess podocytic differentiation in cell lines. Podo- cytes possess a contractile system composed of actin in relation to other proteins, such as myosin IIA and -actinin-4 (reviewed in 10 –13). Synaptopodin is associated with the actin network. The slit diaphragm, the chief size selective filter, is composed of nephrin in association with several other proteins, including CD2-associated protein (CD2AP) and podocin. The main struc- tural protein of the glomerular basement membrane (GBM) is type IV collagen. Type IV collagen is assembled from a family of six distinct chains (reviewed in 14,15), designated 1 to 6. The six type IV collagen chains self-assemble into three basic protomers (each a triple helix) with the composition 1 2 2, 5 2 6, or 345. These protomers then are organized into Received June 10, 2005. Accepted September 28, 2005. Published online ahead of print. Publication date available at www.jasn.org. Address correspondence to: Dr. Paul Scott Thorner, Division of Pathology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Phone: 416-813-5108; Fax: 416-813-5974; E-mail: thorner@sickkids.on.ca Copyright © 2006 by the American Society of Nephrology ISSN: 1046-6673/1701-0066