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. 2015 Sep;9(3):233-46.
doi: 10.1007/s12079-015-0289-3. Epub 2015 Mar 29.

Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines

Payam Ghasemi-Dehkordi  1 , Mehdi Allahbakhshian-Farsani  2 , Narges Abdian  1 , Amin Mirzaeian  1 , Javad Saffari-Chaleshtori  3 , Fatemeh Heybati  1 , Gashtasb Mardani  3 , Alireza Karimi-Taghanaki  1 , Abbas Doosti  4 , Mohammad-Saeid Jami  1 , Marziyeh Abolhasani  1 , Morteza Hashemzadeh-Chaleshtori  5
Affiliations

Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines

Payam Ghasemi-Dehkordi et al. J Cell Commun Signal. 2015 Sep.

Abstract

Human induced pluripotent stem cells (hiPSCs) are a type of pluripotent stem cells artificially derived from an adult somatic cell (typically human fibroblast) by forced expression of specific genes. In recent years, different feeders like inactivated mouse embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and feeder free system have commonly been used for supporting the culture of stem cells in undifferentiated state. In the present work, the culture of hiPSCs and their characterizations on BD Matrigel (feeder-and serum-free system), MEF and HDF feeders using cell culture methods and molecular techniques were evaluated and compared. The isolated HDFs from foreskin samples were reprogrammed to hiPSCs using gene delivery system. Then, the pluripotency ability of hiPSCs cultured on each layer was determined by teratoma formation and immunohistochemical staining. After EBs generation the expression level of three germ layers genes were evaluated by Q-real-time PCR. Also, the cytogenetic stability of hiPSCs cultured on each condition was analyzed by karyotyping and comet assay. Then, the presence of pluripotency antigens were confirmed by Immunocytochemistry (ICC) test and alkaline phosphatase staining. This study were showed culturing of hiPSCs on BD Matrigel, MEF and HDF feeders had normal morphology and could maintain in undifferentiated state for prolonged expansion. The hiPSCs cultured in each system had normal karyotype without any chromosomal abnormalities and the DNA lesions were not observed by comet assay. Moreover, up-regulation in three germ layers genes in cultured hiPSCs on each layer (same to ESCs) compare to normal HDFs were observed (p < 0.05). The findings of the present work were showed in stem cells culturing especially hiPSCs both MEF and HDF feeders as well as feeder free system like Matrigel are proper despite benefits and disadvantages. Although, MEFs is suitable for supporting of stem cell culturing but it can animal pathogens transferring and inducing immune response. Furthermore, HDFs have homologous source with hiPSCs and can be used as feeder instead of MEF but in therapeutic approaches the cells contamination is a problem. So, this study were suggested feeder free culturing of hiPSCs on Matrigel in supplemented media (without using MEF conditioned medium) resolves these problems and could prepare easy applications of hiPSCs in therapeutic approaches of regenerative medicine such as stem-cell therapy and somatic cell nuclear in further researches.

Keywords: BD Matrigel matrix ICC; Comet assay; HDF; IHC; Karyotyping; MEF; hiPSCs.

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Figures

Fig. 1
Fig. 1
a Isolated HDFs from neonatal human foreskin in passage 2, HEK-293T cells transfected with lentiviral plasmid expressing eGFP after 72 h observed in b normal light and c fluorescence, and d HDFs transducing by eGFP lentiviruses after 72 h
Fig. 2
Fig. 2
The cultures of hiPSCs on a MEF feeder, b HDF feeder, and c BD Matrigel matrix (as a feeder free condition)
Fig. 3
Fig. 3
The agarose gel electrophoresis of amplified cDNA samples of pluripotency genes of cultured iPSCs on different layers
Fig. 4
Fig. 4
The ICC test on hiPSC colonies cultured on a BD Matrigel matrix, b MEF and c HDF feeders, and their alkaline phosphatase positive test, respectively
Fig. 5
Fig. 5
The karyotype of hiPSCs cultured on a feeder free system (BD Matrigel), b MEF and c HDF feeders without cytogenetic abnormalities
Fig. 6
Fig. 6
The alkaline comet assay of hiPSCs cultured on a MEF feeder, b HDF feeder, and c feeder free system (BD Matrigel) in low passage compare to d H2O2 group (positive control) and e normal HDFs
Fig. 7
Fig. 7
Histopathology assay of teratomas The teratoma formation of hiPSCs (left) by injection amount of 1 × 106 cells suspended in PBS into dorsal flanks of immunodeficient (SCID) mice and the tissue expansion from teratoma in vivo (right) by H&E staining and immunohistochemical staining (after three times repeated) using human E-cadherin (a-e: three germ layers including neural tissues (ectoderm), gut-like epithelial tissues (endoderm), striated muscle (mesoderm), cartilage (mesoderm), and keratin-containing epidermal tissues (ectoderm), respectively were observed. f-j: IHC staining demonstrates robust expression of human E-cadherin antibody on the surface of each germ layer of generated teratomas)
Fig. 8
Fig. 8
The EB formation from hiPSCs cultured on a MEF feeder, b HDF feeder, and c feeder free system (BD Matrigel matrix)
Fig. 9
Fig. 9
Comparison the mean difference (a 95% confidence interval (CI) were used for the difference) of the expression level of three germ layers genes (mesoderm, ectoderm, and endoderm) in EBs derived from hiPSCs that cultured on each layer, hESCs and HDFs
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