The genetically-modified iPSCs were required to ensure the normal karyotypes, the pluripotent status and the capacity to differentiate into cells of the three germ layers.
Chromosomal analysis was performed by GTG-banding analysis at the Center for Medical Diagnostic Laboratories, Faculty of Medicine, Chulalongkorn University, Thailand, following the recommendations by the International System for Cytogenetics Nomenclature (ISCN).
The pluripotency of stem cells was evaluated by reverse-transcriptase PCR (RT-PCR) of stem cell markers including NANOG, OCT4, SOX2, KLF4 and MYC. The primer sequences are listed in Table1 31 (link)–34 (link).
The ability of stem cells to differentiate into endoderm, mesoderm and ectoderm were tested via embryoid body (EB) formation and stained by Human three germ layer 3-color immunocytochemistry kit (R&D Systems, Minneapolis, MN, USA)35 (link).
To demonstrate the hematopoietic multipotency, our iPSC-derived hematopoietic progenitors were cultured in the presence of 100 ng/ml human thrombopoietin (TPO), 50 ng/ml human stem cell factor (SCF), and 25 ng/ml heparin for 21 days. The culture finally yielded the mixture of megakaryocytes (by morphology and flow cytometry for CD41/CD42b surface expression) and neutrophils (by morphology) as shown in the Supplementary Fig.S8 .
Chromosomal analysis was performed by GTG-banding analysis at the Center for Medical Diagnostic Laboratories, Faculty of Medicine, Chulalongkorn University, Thailand, following the recommendations by the International System for Cytogenetics Nomenclature (ISCN).
The pluripotency of stem cells was evaluated by reverse-transcriptase PCR (RT-PCR) of stem cell markers including NANOG, OCT4, SOX2, KLF4 and MYC. The primer sequences are listed in Table
The ability of stem cells to differentiate into endoderm, mesoderm and ectoderm were tested via embryoid body (EB) formation and stained by Human three germ layer 3-color immunocytochemistry kit (R&D Systems, Minneapolis, MN, USA)35 (link).
To demonstrate the hematopoietic multipotency, our iPSC-derived hematopoietic progenitors were cultured in the presence of 100 ng/ml human thrombopoietin (TPO), 50 ng/ml human stem cell factor (SCF), and 25 ng/ml heparin for 21 days. The culture finally yielded the mixture of megakaryocytes (by morphology and flow cytometry for CD41/CD42b surface expression) and neutrophils (by morphology) as shown in the Supplementary Fig.
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