Reprogramming of peripheral blood from Malaysian beta thalassaemia
patients to induced pluripotent stem cell
-
1
Institute for Medical Research, Stem Cell Laboratory, Haematology Unit, Cancer Research Centre, Malaysia
-
2
Universiti Putra Malaysia, Department of Biomedical Science, Faculty of Medicine and Health Science, Malaysia
-
3
Ampang Hospital, Department of Haematology, Malaysia
Takahashi and Yamanaka first achieved landmark breakthrough by reprogramming mouse embryonic fibroblasts (MEF) into induced pluripotent stem cells (iPSC) via the ectopic expression of only four transcription factors, namely Oct4, Sox2, Klf4 and c-Myc. This process allows for the generation of disease specific iPSC that can be used to model human diseases, and ultimately iPSC may lead to the development of cures for many genetic and degenerative diseases. Βeta (β)-thalassemia major is one of the most common genetic diseases and inherited blood disorder in Malaysia. This disease is characterised by a reduction in the synthesis of haemoglobin subunit β caused by mutation of beta globin gene. Without treatment, affected patients fail to thrive and have a shortened life expectancy. Hence, this research aims to generate iPSC from the peripheral blood of β-thalassaemia patients and characterize the iPSC cells for their potential use in the treatment of the disease. The ethical approval of this study was obtained from Medical Research Ethics Committee, Ministry of Health, Malaysia (project ID:NMRR-14-570-21328). The peripheral blood of three β-thalassaemia major patients was collected fromthe Ampang Hosipital, Kuala Lumpur and reprogrammed to iPSC using sendai virus reprogamming method. Tranduced cellswere resuspended in complete PMBC medium which contained stem cell factor (SCF), FMS-Like Tyrosine Kinase 3 (FLT-3), interleukin 3 and interleukin 6.After about 20 days, colonies resembled embryonic stem cell morphology were picked up with the assistance of live staining marker, Tral-1-60. The iPSC colonies were maintained in Essential 8 medium and further characterized by immunofluorescence staining, RT-PCR and karyotyping.iPSC were also assessed for their differentiation potential including embryoid body formation, in vivo teratoma formation and cardiomyocytes differentiation. In vivo teratoma formation was conducted on SCID mice with approval from the institutional animal ethics committee. Three iPSC clones from β-thalassemia patients were successfully propagated and maintained for this study. The iPSC expressed pluripotent markers such as NANOG, SOX2, OCT 4,Tra-1-60, SSEA4 and free of viral vector as shown in figures.
The derived iPSC also demonstrated similar morphology as compared to human embryonic stem cells (ESC). Hematoxylin and eosin staining of teratoma showed in vivo differentiation into three germ layers including ecoderm, mesoderm and endoderm. The differentiated cardiomyocytes expressed positive result on cardiomyocyte markers.Our study demonstrated a reproducible protocol for reprogramming blood cells by using Sendai virus vector and subsequent differentiation of the iPSC into cardiomyocytes. Genome editing of the derived iPSC would allow the development of autologus cells for the treatment of beta-thalassaemia 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:
Sendai virus,
β-thalassemia,
Peripheral Blood,
induced pluripotent stem cells (IPSC),
Virus Replication
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:
Mohan
TR,
Lim
MN,
Mok
PL,
Ahmad
R,
Satar
J and
Zakaria
Z
(2016). Reprogramming of peripheral blood from Malaysian beta thalassaemia
patients to induced pluripotent stem cell.
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.2016.02.00025
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Received:
08 Dec 2016;
Published Online:
19 Dec 2016.
*
Correspondence:
Mr. Thena R Mohan, Institute for Medical Research, Stem Cell Laboratory, Haematology Unit, Cancer Research Centre, Kuala Lumpur, Selangor, 50588, Malaysia, then1008@gmail.com