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.

Induced pluripotent stem cells (iPSCs) generated from patients with chronic myeloid leukemia (CML) have the potential for disease modelling to study disease pathogenesis and screening therapeutic interventions. In this study, we aimed to generate iPSCs from CD34+ hematopoietic progenitors of CML patients with varying responses to tyrosine kinase inhibitor (TKI) therapy. The generated CML-CD34-iPSC colonies displayed atypical dome-shaped morphology and underwent spontaneous differentiation in a few days. However, supplementation with imatinib (IM), the most widely used TKI to treat CML patients, in the culture medium improved the stability and maintenance of all isolated CML-CD34-iPSC colonies, allowing them to be maintained for more than 20 passages without significant differentiation. In contrast to previous studies, our results indicate that suppressing the BCR::ABL1 oncogenic pathway is essential for efficiently generating stable CML-iPSC colonies. Furthermore, we successfully differentiated these iPSCs to CD34+ hematopoietic progenitors both in the presence and absence of IM. This robust protocol for generating CML-iPSCs provides a valuable resource for disease modelling. The generated iPSCs will be a valuable tool for investigating CML pathophysiology, drug resistance mechanisms, and drug screening to identify novel and effective therapies for this disease.

Diamond Blackfan anemia (DBA) is a congenital hypoplastic anemia characterized by ineffective erythropoiesis. DBA is majorly caused by mutations in the ribosomal protein (RP) genes1. A suitable disease model that yields a continuous supply of erythroid cells is required to study disease pathogenesis and drug discovery. Towards this, we reprogrammed dermal fibroblasts from a DBA patient with a heterozygous mutation c.22-23delAG in the RPS19 gene identified through exome sequencing. To generate induced pluripotent stem cells (iPSCs), we induced episomal expression of the reprogramming factors OTC3/4, L-MYC, LIN28, SOX2, and KLF4, and a p53 shRNA2. The DBA iPSC line CSCRi006-A generated during this study was extensively characterized for its pluripotency and genome stability. The clone retained normal karyotype and showed high expression levels of pluripotency markers, OCT4, NANOG, SOX2, TRA-I-60, TRA-I-81, and SSEA4. It could differentiate into cells originating from all three germ cell layers, as identified by immunostaining for SOX17 (endoderm), Brachyury (mesoderm), and PAX6 (ectoderm). IPSCs provide a renewable source of cells for in vitro disease modeling. CSCRi006-A, a thoroughly characterized iPSC line carrying heterozygous RPS19 c.22-23delAG mutation, is a valuable cell line for the disease modeling of DBA. This iPSC line can be differentiated into different blood cell types to study the mechanisms of disease development and identify potential treatments.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by the BCR-ABL1 fusion gene with constitutively active tyrosine kinase activity. Although the tyrosine kinase inhibitors (TKI) have revolutionized the treatment for CML, withdrawal of TKI therapy in patients on deep molecular response causes disease relapse, primarily due to the persistence of leukemic stem cells (LSCs) that are insensitive to TKI. As LSCs are a rare population of cells, it is challenging to understand the molecular basis of the disease relapse to tailor strategies to eliminate them selectively. Induced pluripotent stem cells (iPSCs) derived from primary CML are increasingly used for disease modeling and high through drug screening. Cryopreserved CD34+ from three chronic phase CML patients (n=3) and CD34+ cells from a normal donor after mobilization were expanded in SFEM II supplemented with CD34+ Expansion Supplement (10X) including UM729 for 3-days and were nucleofected with episomal reprogramming plasmids as described previously (Manian et al., 2018). The nucleofection efficiency was 15% in the expanded CD34+ CML and normal cells. While the normal CD34+ cells formed iPSCs with the characteristic flat morphology between 20-24 days, the CML CD34+ cells formed several dome-shaped colonies after 30 days. Two normal CD34+ iPSC colonies continued to maintain their morphology for 12 passages. All the CML iPSC colonies (n=11) expressed the same type of BCR-ABL1 transcript as the CD34+ cells and did not carry any mutations in the BCR-ABL1 kinase domain. However, only three of the 11 CML-iPSC colonies could be maintained without significant differentiation after five passages. They lacked typical iPSC morphology and appeared as small cell aggregates or dome-shaped colonies. To understand whether this atypical morphology of the CML iPSC colonies was due to the expression of the BCR-ABL fusion protein within the cells, the medium was supplemented with 10μM imatinib. There was an increase in cell proliferation rate and gain of typical iPSCs morphology similar to normal iPSCs within two days after adding IM. Withdrawal of IM from the IM-treated CML iPSC resulted in a dome-shaped morphology, reduced proliferation rate, and reduction in TRA1-60 expression after 96 hrs. The CML iPSC cultured in the presence of IM showed significant downregulation of phospho-CRKL, a BCR-ABL1 downstream signaling protein, compared to the colonies cultured in the absence of IM. These results suggest that suppression of BCR-ABL1 by treatment with IM produced stable CML iPSC-like phenotype while IM withdrawal resulted in dome-shaped colonies and pushed the cells towards differentiation. This proof-of-concept study identified a unique CML LSCs mimetic model. It would serve as an excellent platform for screening small molecules to eliminate LSCs as it thrives on TKI therapy. Citation Format: Esther Benjamin, Gaurav Joshi, Bharathi Rajamani, Krittika Nandy, Shaji Velayudhan, Poonkuzhali Balasubramanian. Imatinib mesylate treatment improves reprogramming efficiency, and morphology of chronic myeloid leukemia derived induced pluripotent stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3153.

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