Identification of FIP200 interaction with the TSC1–TSC2 complex and its role in regulation of cell size control

FIP200 (focal adhesion kinase [FAK] family interacting protein of 200 kD) is a newly identified protein that binds to the kinase domain of FAK and inhibits its kinase activity and associated cellular functions. Here, we identify an interaction between FIP200 and the TSC1–TSC2 complex through FIP200 binding to TSC1. We found that association of FIP200 with the TSC1–TSC2 complex correlated with its ability to increase cell size and up-regulate S6 kinase phosphorylation but was not involved in the regulation of cell cycle progression. Conversely, knockdown of endogenous FIP200 by RNA interference reduced S6 kinase phosphorylation and cell size, which required TSC1 but was independent of FAK. Furthermore, overexpression of FIP200 reduced TSC1–TSC2 complex formation, although knockdown of endogenous FIP200 by RNA interference did not affect TSC1–TSC2 complex formation. Lastly, we showed that FIP200 is important in nutrient stimulation-induced, but not energy- or serum-induced, S6 kinase activation. Together, these results suggest a cellular function of FIP200 in the regulation of cell size by interaction with the TSC1–TSC2 complex

Synthetic surface for expansion of human mesenchymal stem cells in xeno-free, chemically defined culture conditions.

Human mesenchymal stem cells (HMSCS) possess three properties of great interest for the development of cell therapies and tissue engineering: multilineage differentiation, immunomodulation, and production of trophic factors. Efficient ex vivo expansion of hMSCs is a challenging requirement for large scale production of clinical grade cells. Low-cost, robust, scalable culture methods using chemically defined materials need to be developed to address this need. This study describes the use of a xeno-free synthetic peptide acrylate surface, the Corning® Synthemax® Surface, for culture of hMSCs in serum-free, defined medium. Cell performance on the Corning Synthemax Surface was compared to cells cultured on biological extracellular matrix (ECM) coatings in xeno-free defined medium and in traditional conditions on tissue culture treated (TCT) plastic in fetal bovine serum (FBS) supplemented medium. Our results show successful maintenance of hMSCs on Corning Synthemax Surface for eight passages, with cell expansion rate comparable to cells cultured on ECM and significantly higher than for cells in TCT/FBS condition. Importantly, on the Corning Synthemax Surface, cells maintained elongated, spindle-like morphology, typical hMSC marker profile and in vitro multilineage differentiation potential. We believe the Corning Synthemax Surface, in combination with defined media, provides a complete synthetic, xeno-free, cell culture system for scalable production of hMSCs

Defined culture of human embryonic stem cells and xeno-free derivation of retinal pigmented epithelial cells on a novel, synthetic substrate.

Age-related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno-free) are preferred for clinical translation. This avoids exposing AMD patients to animal-derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II-SC, which is a novel, synthetic animal-derived component-free, RGD peptide-containing copolymer compliant with good manufacturing practices designed for xeno-free stem cell culture. Cells on Synthemax II-SC were compared with cultures grown with xenogeneic and xeno-free control substrates. This report demonstrates that Synthemax II-SC supports long-term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE-specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium-derived factor. Both hESCs and hESC-RPE maintained normal karyotypes after long-term culture on Synthemax II-SC. Furthermore, RPE generated on Synthemax II-SC are functional when seeded onto parylene-C scaffolds designed for clinical use. These experiments suggest that Synthemax II-SC is a suitable, defined substrate for hESC culture and the xeno-free derivation of RPE for cellular therapies

Defined culture of human embryonic stem cells and xeno-free derivation of retinal pigmented epithelial cells on a novel, synthetic substrate.

Age-related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno-free) are preferred for clinical translation. This avoids exposing AMD patients to animal-derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II-SC, which is a novel, synthetic animal-derived component-free, RGD peptide-containing copolymer compliant with good manufacturing practices designed for xeno-free stem cell culture. Cells on Synthemax II-SC were compared with cultures grown with xenogeneic and xeno-free control substrates. This report demonstrates that Synthemax II-SC supports long-term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE-specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium-derived factor. Both hESCs and hESC-RPE maintained normal karyotypes after long-term culture on Synthemax II-SC. Furthermore, RPE generated on Synthemax II-SC are functional when seeded onto parylene-C scaffolds designed for clinical use. These experiments suggest that Synthemax II-SC is a suitable, defined substrate for hESC culture and the xeno-free derivation of RPE for cellular therapies

Long-term multipassage culture of hBM-MSCs in different surface/media conditions.

<p>Cells were cultured for 8 sequential passages using the indicated conditions. Seeding density at each passage was 7000 cells/cm². Cumulative cell number (A), doubling time (B), cumulative population doublings (PDs) (C), and % viability (D) across the multiple passages are shown. The error bars in D represent the average +/− STDEV from 7 serial passages. Statistical analyses by an ANOVA followed by the Tukey’s method performed on the doubling time (7 passages) and % viability (8 passages) found that doubling time of TCT/with serum was significantly higher and that the % viability for TCT/with serum was significantly lower compared to the ECM and Synthemax Surface serum-free conditions (p-value = 0.000).</p

Long-term culture of hAD-MSCs in different surface/media conditions.

<p>Cells were cultured for 3 sequential passages in T75 flasks using the indicated conditions. Seeding density at each passage was 5000 cells/cm². Phase contrast micrographs with representative cell morphology on day 6 (A) cumulative cell number (B), doubling time ± STDEV (C), cumulative population doublings (PDs) (D), and % viability ± STDEV (E) across 3 passages, are shown for the indicated conditions.</p

Trilineage differentiation of hBM-MSC after long-term culture on Corning® Synthemax® Surface in xeno-free defined medium.

<p>Cells were cultured on Corning Synthemax Surface for 9 passages prior to the induction of adipogenic, osteogenic, and chondrogenic differentiations. (A) Oil Red O staining of lipid droplets after 20 days of adipogenic induction, (B) Alizarin Red staining of calcium phosphate deposits produced by osteocytes after 21 days of osteogenic induction, (C) Alcian Blue staining of proteoglycans synthesized by chondrocytes after 14 days of chondrogenic induction.</p

Attachment and short term growth of hBM-MSCs in different surface/media conditions.

<p>7000 cells/cm² were seeded in 6 well plates using the indicated conditions. Cells were harvested after 24 (A) and 96 (B) hours to determine the plating efficiency and cell expansion, respectively. The dotted line in (B) indicates the seeding density. The error bars in A and B represent the average +/− STDEV from 2 (panel A) and 5 (panel B) independent experiments. Statistical analyses by an ANOVA followed with a Tukey’s method performed on the viable cells per cm² (B) found that the samples are statistically different from each other (p-value = 0.000) and that the ECM (**) is higher than both the Synthemax and TCT/serum, and Synthemax (*) is higher than TCT/serum condition. (C) The representative cell morphology micrographs for the indicated surface/media conditions. Scale bar is 100 µm.</p

hBM-MSC culture on Corning® Synthemax® Surface in different commercially available serum-free, defined media.

<p>Cells were seeded at the density of 3500 cells/cm² in 6 well plates with Corning Synthemax Surface in the indicated media and allowed to expand to 80–90% confluence prior to harvest. (A) Phase contrast micrographs show cell morphology at 48 hours and prior to harvest for different media. (B) Doubling time for cells cultured on Corning Synthemax Surface in different media condition for one passage (3–7 days depending on the media).</p