Abstract
Aims
Down syndrome (DS) is the most frequent single cause of human birth defects and intellectual disability (ID). Its etiology has been known for over 50 years, DS is caused by trisomy of chromosome 21 (Ts21). In addition, there are more evidences in clinical show that patients of DS are also have high incidence of leukemia, but its underlying mechanisms have yet to be discovered. Elucidation of these mechanisms has been hindered by the difficulties in isolating and expanding enough hematopoietic stem cells (HSCs) from the patients. Circular RNAs (CircRNAs) are a novel type of endogenous noncoding RNAs that reported to play important roles in biological and pathological processes. Our present study aims to investigate the pathogenesis of Down Syndrome prone to leukemia using iPSCs from DS patients and the modulation mechansim of key target gene and circRNAs.
Methods
To overcome the limitation of clinical sample resources, we developed Ts21-induced pluripotent stem cells (iPSCs) from mononuclear cells using Episomal vectors which highly express Oct4,Sox2 and Klf4. Under certain conditions, these Ts-21 specific iPSCs could be further induced into any cell types, including HSCs. Next, we induced the patient-specific iPSCs differentiate into HSCs under serum free and feeder-free condition in vitro. By using flow cytometry, qRT-PCR and CFU assays, we have made well comparisons between HSCs derived from TS-21 iPSCs and control iPSCs, and expolre the pathological feature and mechanisms. By performing circRNA-seq analysis, we identified key circRNA and its host gene regulatory networks in DS with hematopoietic disorder. On the basis of that, we can further search for the potential drug targets that effective against leukemia of DS patients.
Results
We show here by gene sequencing, karyotyping and immunohistochemistry staining, the initial characterization of the patient-specific induced pluripotent stem are confirmed to be fully reprogrammed and they still carry the original mutation. These cells can be used for screening approaches and can be modified for loss- and gain-of-function strategies to identify pathways and test candidate mechanisms. We have observed that myeloid differentiation potential of HSCs derived from DS patients was significantly higher than normal patinets. DS-iPSCs exhibited a 2-4 fold increase in a population of CD43+/GPA+ hematopoietic cells, accompanied by increased multilineage colony-forming potential in CFU assays.During this process, has_circ_0082802 was significantly up-regulated. After knockdown of has_circ_0082802 using shRNAs, the erythroid differentiation level as well as cell proliferation were decreased. The potential modulation mechansim of them will be further explored.
Conclusion
We established the cellular models of DS patients and derived differentiation into different cell types in vitro, which has the similar function with patient HSCs. On the basis of that, we found has_circ_0082802 may play important role in erythroid differentiation. We can further search for the potential drug targets that
effective against leukemia of DS patients. This approach would provide a powerful cell resource for clinical research and a useful model for the study of the mechanisms of DS-AML
Disclosures
No relevant conflicts of interest to declare.
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