Neuropotency of Human Mesenchymal Stem Cell Cultures: Clonal Studies Reveal the Contribution of Cell Plasticity and Cell Contamination Rodrigo Somoza,

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Neuropotency of Human Mesenchymal Stem Cell Cultures: Clonal Studies Reveal the Contribution of Cell Plasticity and Cell Contamination Rodrigo Somoza, Paulette Conget, Francisco Javier Rubio Biology of Blood and Marrow Transplantation Volume 14, Issue 5, Pages 546-555 (May 2008) DOI: 10.1016/j.bbmt.2008.02.017 Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Real-time PCR analysis for mRNA detection. (A) Melting temperature (Tm) for amplicons was calculated from peaks obtained from –d[F]/dT versus T plots (F = SYBR Green fluorescence; T = °C) using a LightCycler® thermocycler (n = 3-5 reactions). Theorical amplicon size was verified by DNA-electrophoretic analysis (showed on the left side of each melting curve). Graphics at the botton of part A show representatives fluorescent-amplification profiles and a standard curve for GAPDH used to determinate the detection limit for PCR product. (B) Sequences of primers used and characteristic of the amplicons. n.d.: not determinated Biology of Blood and Marrow Transplantation 2008 14, 546-555DOI: (10.1016/j.bbmt.2008.02.017) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Cellular and molecular characterization of hMSC cultures. (A) Cells showed a fibroblastic-like morphology and (B) immunolabeled using FITC-conjugated anti-ASMA monoclonal antibody. (C) hMSC culture-derived cells differentiate into adipocytes, (D) osteocytes, and (E) chondrocytes. (F) Gene expression profile of hMSC cultures using RT-PCR assay. +, detected; −, nondetected; +/−, detected in some samples. Scale bars: 100 μm. Biology of Blood and Marrow Transplantation 2008 14, 546-555DOI: (10.1016/j.bbmt.2008.02.017) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Mesenchymal potential of four representative hMSC culture-derived clones. Adipogenesis assessed by accumulation of lipid vacuoles (Oil Red O), osteogenesis indicated by extracellular calcium salts deposition (Alizarin Red). AdP: adipogenic precursors; OsP: osteogenic precursors. Scale bars: 100 μm. Biology of Blood and Marrow Transplantation 2008 14, 546-555DOI: (10.1016/j.bbmt.2008.02.017) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Phenotypical changes in hMSC culture-derived clones after applying the neural protocol. (A) Photomicrographs showed no differences in the adherence to plastic and cell morphology between representative mesenchymal (B10 and A08) and nonmesenchymal (C02) clones before exposure to the neural protocol (d0), although there were differences in the neural gene expression pattern. Floating and adherent neurosphere-like structures and mature neural-like cells appear after applying the neural protocol (d8). (B) Typical morphology of rNSC culture-derived cells in different states of differentiation. Scale bars: 50 μm. Biology of Blood and Marrow Transplantation 2008 14, 546-555DOI: (10.1016/j.bbmt.2008.02.017) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Mechanisms to explain neuropotency in hMSC culture-derived clones. (A) Distinct neural gene expression patterns found in clones before and after applying the neural protocol are represented in this chart. (B,C) Mesenchymal clones showed neural cell plasticity given by the mecanisms of transdifferentiation (B, 11 clones) and pluripotency (C, 15 clones). (D) The existence of 2 nonmesenchymal clones with neuropotency indicates neural cell contamination. Arrows represent the theoretical gene expression-routes of each clone. Beginning and end of arrows are related to neural gene expression patterns before and after the application of the neural protocol, respectively; dotted rectangles indicate nondifferentiated clones; black and white arrows indicate clones maturing and dedifferentiating, respectively. Biology of Blood and Marrow Transplantation 2008 14, 546-555DOI: (10.1016/j.bbmt.2008.02.017) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions