Generation of neural stem cells from human peripheral blood derived induced pluripotent stem cells
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1
Institute for Medical Research, Stem Cell Laboratory, Haematology Unit, Cancer Research Centre, Malaysia
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2
Universiti Putra Malaysia, Department of Biomedical Science, Faculty of Medicine and Health Science, Malaysia
Induced pluripotent stem cells (iPSC) have recently evolved as a potential cell source for disease modelling and therapy. However, most studies have targeted skin fibroblasts instead of easily accessible source such as peripheral blood for reprogramming. In order to harness the cell reprogramming technology, we produced iPSC lines from human peripheral blood samples and evaluated the differentiation potential of the derived iPSC to neuronal stem cells.
The ethical approval for conducting this study was obtained from the Medical Research Ethical Committee, Ministry of Health, Malaysia (project ID: NMRR-14-570-21328). Two mL of peripheral blood from three normal donors were collected and the mononuclear cells of the samples were reprogrammed with Sendai virus vectors carrying Yamanaka factors. The transduced cells were plated and cultured in complete medium supplemented with stem cell factor (SCF), FMS-Like Tyrosine Kinase 3 (FLT-3), interleukin 3 and interleukin 6 before transferring to mouse embryonic fibroblast culture dishes. Emergence of iPSC colonies were monitored and picked with Tra-1-60 live staining method. The best iPSC clone from each donor was maintained in feeder free media. The iPSC clones were then characterized by immunofluorescence staining, flow cytometry, microarray analysis, RT-PCR, embryoid bodies formation and karyotyping. In addition, in vivo teratoma formation assay was also performed using SCID mice. In brief, the blood-derived iPSC were injected subcutaneously into SCID mice and allowed to form teratoma which was about 1cm in diameter. The mice were then eutinized and tumour was collected for histopathology analysis with haematoxylin and eosin staining. The blood-derived iPSC resembled embryonic stem cells (ESC) morphology with flatter cobblestone-like appearance and prominent nucleus (Figure 1a). These iPSC expressed pluripotent markers such as OCT 3/4, SOX2, NANOG, SSEA4, and have similar gene expression profile as compared to the control ESC (H9) and iPSC (IMR-90) (Figure 1b). Besides showing normal karyotype, the cells were free of Sendai virus by RT-PCR analysis (Figure 1b) and formed embryoid bodies and teratomas (Figure 1c). Upon neural differentiation using neural induction medium and neuronal expansion medium, the iPSC showed neural stem cell morphology and expressed neural stem cell markers such as Nestin and Pax6 (Figure 2).
Our data shows that blood derived iPSC were successfully generated. To our knowledge, this is the first blood-derived iPSC generated in this country. The derived iPSC not only transgene-free, but possessed differentiation potential towards neural stem cells and could be used for neuron cells generation in the future.
Acknowledgements
The authors wish to thank the Director General of Health, Malaysia for permission to publish this paper. This study was supported by a Ministry of Health grant, NMRR-14-570-21328/ JPP-IMR: 14–024.
Keywords:
Blood,
Sendai virus,
neural stem cell,
Pluripotent markers,
induced pluripotent stem cells (IPSC)
Conference:
6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting, Seberang Jaya, Penang, Malaysia, 17 Nov - 18 Nov, 2016.
Presentation Type:
Poster
Topic:
Gene Therapy, Reprogramming and Pluripotency
Citation:
Ramachandran
S,
Lim
MN,
Mok
PL,
Fakiruddin
KS,
Fakharuzi
NA and
Zakaria
Z
(2016). Generation of neural stem cells from human peripheral blood derived induced pluripotent stem cells.
Front. Bioeng. Biotechnol.
Conference Abstract:
6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting.
doi: 10.3389/conf.FBIOE.2016.02.00022
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Received:
08 Dec 2016;
Published Online:
19 Dec 2016.