These results clearly indicate our system not merely allows the maintenance of personal renewal but, more importantly, promotes pluripotency. Na?ve or Primed To further assess the quality of stem cells cultured in NM23-H1-MM on anti-MUC1* antibody surfaces, we measured expression levels of a subset of genes that are thought to be indicators of human stem cells being in the na?ve or ground state. were previously purified by size exclusion chromatography.(TIF) pone.0058601.s002.tif (74K) GUID:?274D8E22-13A8-4C9E-84CC-1BB1C5848499 Figure S3: The addition of recombinant NM23 to NM23-depleted conditioned media eliminates the need for added bFGF. a) hES cells on Matrigel grew pluripotently in standard bFGF plus conditioned media from human HS27 feeder cells (control); b) The same cells were cultured in bFGF plus HS27 conditioned media that had been immuno-depleted of NM23 and cells immediately differentiated. c) Cells cultured in bFGF plus depleted conditioned media that had been reconstituted with recombinant NM23 grew pluripotently and indistinguishably from your control. d) Cells cultured in absence of bFGF in depleted conditioned media that had been reconstituted with recombinant NM23 grew as well as the control showing that the requirement for bFGF is usually eliminated by addition of recombinant NM23.(TIF) pone.0058601.s003.tif (968K) GUID:?CC324BC8-67F9-4E4C-AC7F-EF962B35D583 Figure S4: The stability of NM23S120G-dimer under culture conditions was tested. NM23S120G-dimer was added to cell culture media and kept in a CO2 incubator for up to 48 hours, then analyzed by western blot, which showed that no denaturation occurred within the time frame required for use in stem cell culture.(TIF) pone.0058601.s004.tif (247K) GUID:?B6F8BDEC-B166-4EC5-A06F-54D66AAB3672 Physique S5: Withdrawal of growth factor NM23-H1 S120G-dimer and inhibition of NM23-H1-MUC1* interaction induce differentiation. EPZ020411 hydrochloride H9 hES cells were cultured in either bFGF plus conditioned media or in NM23-H1S120G-dimer, and then allowed to differentiate by withholding the growth factor (a-d and e-h, respectively). Some cells also received the MUC1*ecd peptide (1 M) to competitively EPZ020411 hydrochloride inhibit the NM23-H1-MUC1* conversation (iCj). Withdrawing the growth factor bFGF or in NM23-H1S120G-dimer EPZ020411 hydrochloride induces differentiation with a maximum at 144 h. However, blocking the conversation between in NM23-H1 and MUC1* prematurely induces differentiation (96 h).(TIF) pone.0058601.s005.tif (3.8M) GUID:?393F1CD1-4142-468E-AC51-71350BCFF45D Physique S6: ES and iPS cells cultured in NM23-MM grow comparably to cells cultured in bFGF as assessed by cell morphology. a, b) Human H9ES cells cultured on MEFs in either NM23-MM or bFGF both appear to grow as undifferentiated stem cell colonies. c, d) H9s on Matrigel that were cultured in either NM23-MM or bFGF plus MEF conditioned media appear to grow comparably as pluripotent colonies. e, f) iPS cells cultured in NM23-MM on MEFs grew faster than the same cell collection cultured in bFGF. g) iPS cells grew as well on Matrigel as they had on MEFs. All photos at 4X magnification.(TIF) pone.0058601.s006.tif (6.5M) GUID:?117B9ACA-06E5-4F60-AC95-B6612FE63F5E Physique S7: hES and iPS cells karyotypes. H9s and iPS on Matrigel that had been serially passaged at least six (6) occasions had normal karyotype (a and b). H9s and iPS cells on a monoclonal anti-MUC1* antibody (MN-C3) surface that had been serially EPZ020411 hydrochloride passaged at least six (6) occasions had normal karyotype (c and d).(TIF) pone.0058601.s007.tif (748K) GUID:?CB905536-5B18-46EF-A02C-2D5DE70A91BA Physique S8: Quantification, by fow cytometry, of the pluripotency markers expressed around the cell surface of human stem cell cultured in NM23-H1-MM over anti-MUC1* antibody surfaces. a and d) The pluripotency markers Tra 1-60 (a), SSEA-4 (a) and SSEA-3 (b) are expressed around the cell surface. c) The differenciation marker CXCR4 is usually barely expressed around the cell surface. d) percentage of cells expressing the different markers tested.(TIF) pone.0058601.s008.tif (1.1M) GUID:?27BD9E1A-3999-48C0-83EC-9765AC95FAE8 Figure S9: iPS, H14, H7 and H9 cells cultured in NM23-H1-MM on anti-MUC1* surfaces express essentially the same or higher levels of the EPZ020411 hydrochloride pluripotency genes than cells cultured in bFGF on MEFs. a) A number of stem cells were cultured in either bFGF over MEFs or NM23-H1-MM over anti-MUC1* antibody MN-C3 surfaces for 10C12 passages, then assayed by RT-PCR to measure expression levels of pluripotency genes Oct4, Nanog, Klf4, and Klf2 Rabbit Polyclonal to TACC1 and miR-145, an indication of the cell’s exit from pluripotency. Growth in NM23-H1-MM on anti-MUC1* ab surfaces maintains pluripotency over multiple passages for several cell lines with the same or increased expression of.