Low efficiency of somatic cell reprogramming and heterogeneity among human induced pluripotent stem cells (hiPSCs) demand extensive characterization of isolated clones before their use in downstream applications. By monitoring human fibroblasts undergoing reprogramming for their morphological changes and expression of fibroblast (CD13), pluripotency markers (SSEA-4 and TRA-1-60) and a retrovirally expressed red fluorescent protein (RV-RFP), we compared the efficiency of these features to identify bona fide hiPSC colonies. The co-expression kinetics of fibroblast and pluripotency markers in the cells being reprogrammed and the emerging colonies revealed the heterogeneity within SSEA-4+ and TRA-1-60+ cells, and the inadequacy of these commonly used pluripotency markers for the identification of bona fide hiPSC colonies. The characteristic morphological changes in the emerging hiPSC colonies derived from fibroblasts expressing RV-RFP showed a good correlation between hiPSC morphology acquisition and silencing of RV-RFP and facilitated the easy identification of hiPSCs. The kinetics of retroviral silencing and pluripotency marker expression in emerging colonies suggested that combining both these markers could demarcate the stages of reprogramming with better precision than with pluripotency markers alone. Our results clearly demonstrate that the pluripotency markers that are routinely analyzed for the characterization of established iPSC colonies are not suitable for the isolation of pluripotent cells in the early stages of reprogramming, and silencing of retrovirally expressed reporter genes helps in the identification of colonies that have attained a pluripotent state and the morphology of human embryonic stem cells (hESCs).

Reliable human erythroid progenitor cell (EPC) lines that can differentiate to the later stages of erythropoiesis are important cellular models for studying molecular mechanisms of human erythropoiesis in normal and pathological conditions. Two immortalized erythroid progenitor cells (iEPCs), HUDEP-2 and BEL-A, generated from CD34+ hematopoietic progenitors by the doxycycline (dox) inducible expression of human papillomavirus E6 and E7 (HEE) genes, are currently being used extensively to study transcriptional regulation of human erythropoiesis and identify novel therapeutic targets for red cell diseases. However, the generation of iEPCs from the patients with red cell diseases is challenging as obtaining a sufficient number of CD34+ cells require bone marrow aspiration or their mobilization to peripheral blood using drugs. This study established a protocol for culturing early-stage EPCs from peripheral blood (PB) and their immortalization by expressing HEE genes. We generated two iEPCs, PBiEPC-1 and PBiEPC-2, from the peripheral blood mononuclear cells (PBMNCs) of two healthy donors. These cell lines showed stable doubling times with the properties of erythroid progenitors. PBiEPC-1 showed robust terminal differentiation with high enucleation efficiency, and it could be successfully gene manipulated by gene knockdown and knockout strategies with high efficiencies, without affecting its differentiation. This protocol is suitable for generating a bank of iEPCs from patients with rare red cell genetic disorders for studying disease mechanisms and drug discovery.

Generation of Fanconi anemia (FA) patient-specific induced pluripotent stem cells (iPSCs) has been reported to be technically challenging due to the defects in the FA-pathway in the patients' somatic cells. By inducible complementation of FA-pathway, we successfully reprogrammed the fibroblasts of an FA patient to iPSCs. CSCR19i-indCFANCA, one of the iPSC lines generated by the inducible complementation of FA-pathway, was extensively characterized for its pluripotency and karyotype. In the absence of doxycycline (DOX) and FANCA expression, this line showed the cellular phenotypes of FA, suggesting it is an excellent tool for FA disease modeling and drug screening.

Fanconi anemia (FA) is an inherited bone marrow failure syndrome caused by a defect in one of the DNA repair pathways. FA exhibits a high degree of clinical heterogeneity and the exact molecular basis of the difference in the phenotypes has not been well understood. Study of this disease in geographic regions with high consanguinity will provide valuable insights in genotype-phenotype correlation and the genetic and environmental modifiers of this disease. We carried out a comprehensive clinical and molecular analysis in 101 patients with pancytopenia, with age <20 years, seen at the department of Haematology, Christian Medical College, India between 2009 and 2014. Seventy six patients had characteristic physical abnormalities of FA, of which perioral hyperpigmentation (42%) was the most common in these patients. We also included 25 patients with aplastic anemia as controls. The median age at diagnosis was 11 years (range 4-30) and sex ratio between males to females was 3:2. Chromosome breakage analysis (CBA) was performed on peripheral blood samples at diagnosis. Forty metaphases each from patient and normal control were assessed for sensitivity to Mitomycin C as per the protocol published in Mayo Clinic Proceedings (1997). CBA score greater than the cut off value of 40 was seen in 63% (n=48/76) of the patients. However, 23% (n=18/76) had ambiguous or borderline score (score 30-40; n= 12, score 20-30; n=4, score <10-20; n=2) and 13% (n=10/76) of patients were not sensitive to Mitomycin C. The control group did not display sensitivity or had mild degree of breakage (CBA score: 4.1-20). FANCD2 ubiquitination analysis of these patients with physical abnormalities in peripheral blood and fibroblasts showed absence of ubiquitination in 69 (90%) of the patients while the control group had normal ubiquitination pattern. To evaluate mosaicism, both peripheral blood and fibroblast samples were analyzed in 59 patients and FANCD2 ubiquitination was defective in both tissues in 55 (93%) patients. In 3 patients, FANCD2 ubiquitination was defective in fibroblasts, but not in peripheral blood indicative of mosaicism of this disease in these patients. Three patients had a normal ubiquitination pattern but had high CBA score and this might be due to mutations in any of the genes that function in the pathway downstream of FANCD2 ubiquitination and requires further evaluation. Four patients who had physical features suggestive of FA were negative by both CBA and FANCD2 western blot. In the control group, both peripheral blood and fibroblast samples were available in 19 patients all of which showed normal FANCD2 ubiquitination. In order to further characterize the FA subtypes, we generated lentiviral vectors to express FANCA, FANCC and FANCG genes for complementation assay. Fibroblasts from 20 patients were transduced with lentiviral FANCA and in 17 (85%) FANCD2 ubiquitination could be restored suggesting a high frequency of FANCA defects in Indian population. Mutation screening of FANCA in a subset of patients showed large deletions in five of which three are novel (ex 10_37 del,ex 6_14 del and ex 1_4 del). There was a deletion of two nucleotides resulting in a frameshift mutation in one patient (c.3760_3761delGA) and a missense mutation (c.2786A>C) in another. Our data suggests that FANCD2 ubiquitination analysis in conjunction with CBA is useful for the diagnosis of FA and detection of mosaicism and the complementation analysis shows high frequency of FANCA defects in patients with FA in Indian population. Disclosures No relevant conflicts of interest to declare.