Novel autogenic feeders derived from human embryonic stem cells (hESCs) support an undifferentiated status of hESCs in xeno-free culture conditions

Chen, H.-F.; Chuang, Ching Yu; Shieh, Yu Kai; Chang, Hao Wei; Ho, Hong Nerng; Kuo, Hung-Chih

doi:10.1093/humrep/dep003

Cited by 35 publications

(25 citation statements)

References 35 publications

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“…The protocols of pluripotent stem cell culture were modified from previous reports (8)(9)(10)29). Several pluripotent stem cell lines were used, including NTU1 hESCs (karyotype 46, XX) derived by us (10), H9 hESCs (karyotype 46, XX; WiCell, WI, USA) (35), and hiPSCs derived by us (iGra1, iGra2, iDPC3, iCFB46, and iCFB50; see below).…”

Section: Pluripotent Stem Cell Culture Differentiation and Treatmentmentioning

confidence: 99%

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Characteristic Expression of Major Histocompatibility Complex and Immune Privilege Genes in Human Pluripotent Stem Cells and Their Derivatives

Chen

et al. 2015

Cell Transplant

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Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have been regarded as useful sources for cell-based transplantation therapy. However, immunogenicity of the cells remains the major determinant for successful clinical application. We report the examination of several hESC lines (NTU1 and H9), hiPSC lines, and their derivatives (including stem cell-derived hepatocytes) for the expression of major histocompatibility complex (MHC), natural killer (NK) cell receptor (NKp30, NKp44, NKp46) ligand, immune-related genes, human leukocyte antigen (HLA) haplotyping, and the effects in functional mixed lymphocyte reaction (MLR). Flow cytometry showed lower levels (percentages and fluorescence intensities) of MHC class I (MHC-I) molecules, b2-microglobulin, and HLA-E in undifferentiated stem cells. The levels were increased after cotreatment with interferon-g and/or in vitro differentiation. Antigen-presenting cell markers (CD11c, CD80, and CD86) and MHC-II (HLA-DP, -DQ, and -DR) remained low throughout the treatments. Recognition of stem cells/derivatives by NK lysis receptors were lower or absent. Activation of responder lymphocytes was significantly lower by undifferentiated stem cells than by allogeneic lymphocytes in MLR, but differentiated NTU1 hESCs induced a cell number-dependent lymphocyte proliferation comparable with that by allogeneic lymphocytes. Interestingly, activation of lymphocytes by differentiated hiPSCs or H9 cells became blunted at higher cell numbers. Real-time reverse transcriptase PCR (RT-PCR) showed significant differential expression of immune privilege genes Arginase 2, Indole 1, GATA3, POMC, VIP, CALCA, CALCB, CD95L, CR1L, Serpine 1, HMOX1, IL6, LGALS3, HEBP1, THBS1, CD59, and LGALS1) in pluripotent stem cells/derivatives when compared to somatic cells. It was concluded that pluripotent stem cells/derivatives are predicted to be immunogenic, though evidence suggests some level of potential immune privilege. In addition, differential immunogenicity may exist between different pluripotent stem cell lines and their derivatives.

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Section: Pluripotent Stem Cell Culture Differentiation and Treatmentmentioning

confidence: 99%

“…The MHC profiling (MHC class I, II, and nonclassical MHC molecules) of pluripotent stem cells was analyzed by flow cytometry using modified protocols from a previous report (8). All the primary antibodies were used at 1:10 dilutions, and secondary antibodies were used at 1:200 dilutions.…”

Section: Flow Cytometry and Hla Haplotypingmentioning

confidence: 99%

Characteristic Expression of Major Histocompatibility Complex and Immune Privilege Genes in Human Pluripotent Stem Cells and Their Derivatives

Chen

et al. 2015

Cell Transplant

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“…Sperms were washed twice in fertilization (FERT-TALP) medium containing heparin (10 μg/ml), fatty acids free BSA (6 mg/ml) and sodium pyruvate (0.25 mM) by centrifugation at 850 g for 10 min each. Sperm motility and concentration (1∼2×10 6 ) were assessed with a hemocytometer. In vitro matured oocytes were washed in TALP medium and incubated with 70 μl droplets of sperm suspension (15∼20 oocytes/droplet).…”

Section: In Vitro Embryo Productionmentioning

confidence: 99%

“…However, still various types of cells like: fibroblasts (1,5), granulosa (6) and oviductal (7) as feeder layer have been using for the culture and establishment of ESCs in various animal species. Moreover, use of somatic cell as feeder layers limits the stem cell research design, since experimental data may result from a combined ESC and feeder cell response to various stimuli.…”

Section: Introductionmentioning

confidence: 99%

Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices

Sharma

Dubey²,

Kumar³

et al. 2013

Int J Stem Cells

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Background and Objectives: Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. Methods and Results: Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA-4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. Conclusions: Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.

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“…Under more circumstances, they are plated directly on feeder layers originated from MEFs (Thomson et al 1998;Yu et al 2009;Hu et al 2011), STO cells (Park et al 2004;Takahashi and Yamanaka 2006), human embryonic fibroblasts (Kibschull et al 2011), human placental fibroblasts (Genbacev et al 2005), human foreskin fibroblasts (Unger et al 2009;Skottman 2010;Strom et al 2010), human skin fibroblasts (Richards et al 2003;Tecirlioglu et al 2010), amniotic mesenchymal cells (Zhang et al 2011), etc. Human embryonic stem cells (hESCs) can even be cultured on feeder layers derived from themselves (Xu et al 2004;Stojkovic et al 2005;Chen et al 2009). The usage of feeder layers is mainly based on the knowledge listed as follows: ESCs can get sufficient nutrition from blood or via intercellular exchange in vivo.…”

Section: Introductionmentioning

confidence: 99%

A reliable and economical method for gaining mouse embryonic fibroblasts capable of preparing feeder layers

et al. 2015

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Mouse embryonic fibroblasts (MEFs) are widely used to prepare feeder layers for culturing embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in vitro. Transportation lesions and exorbitant prices make the commercially obtained MEFs unsuitable for long term research. The aim of present study is to establish a method, which enables researchers to gain MEFs from mice and establish feeder layers by themselves in ordinary laboratories. MEFs were isolated from ICR mouse embryos at 12.5-17.5 day post-coitum (DPC) and cultured in vitro. At P2-P7, the cells were inactivated with mitomycin C or by X-ray irradiation. Then they were used to prepare feeder layers. The key factors of the whole protocol were analyzed to determine the optimal conditions for the method. The results revealed MEFs isolated at 12.5-13.5 DPC, and cultured to P3 were the best choice for feeder preparation, those P2 and P4-P5 MEFs were also suitable for the purpose. The P3-P5 MEFs treated with 10

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Novel autogenic feeders derived from human embryonic stem cells (hESCs) support an undifferentiated status of hESCs in xeno-free culture conditions

Abstract: Novel autogenic feeders can be derived from hESCs under xeno-free conditions and they can robustly maintain the pluripotent identity of hESCs in xeno-free media containing a low concentration of HS.

Cited by 35 publications

References 35 publications

Characteristic Expression of Major Histocompatibility Complex and Immune Privilege Genes in Human Pluripotent Stem Cells and Their Derivatives

Characteristic Expression of Major Histocompatibility Complex and Immune Privilege Genes in Human Pluripotent Stem Cells and Their Derivatives

Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices

A reliable and economical method for gaining mouse embryonic fibroblasts capable of preparing feeder layers

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