Abstract
Differentiated cells can be reprogrammed to embryonic stem cell-like cells called induced pluripotent stem cells (iPSCs), in which the natural developmental differentiation process is reversed. It is unclear whether the multi-lineage cells can be isolated and identified during reprogramming. In the current study, we detected the expression of lineage markers, isolated neural lineages, and identified the related microRNAs during iPSC formation. Our results demonstrated that a neuroectoderm appeared earlier than mesoderm and definitive endoderm before forming colonies when mouse embryonic fibroblasts were subjected to iPSC formation using transcription factors (TFs). On day 3, the cells expressed Sox1 and Nestin and had ultrastructure consistent with the transition to identity neural germ layer lineage. Fluorescence-activated cell sorting analysis revealed a peak (40%) in neural progenitor marker–positive cells. When subsequently cultured in a neural precursor cell medium, these cells proliferated slowly, became round and aggregated, generating into neurons and glia. Genome-wide microRNA (miRNA) analysis identified 45 differentially regulated miRNAs. Molecular network analysis demonstrated that these miRNAs validated 6,047 experimental mRNA targets. The GO functional annotation analysis of mRNA targets revealed that most genes were related to neurogenesis, such as growth cone, neuronal cell body, neuron projection, and cell junction synapse. The network of protein–protein interactions was observed, which demonstrated that key nodes of neural lineage reprogramming-associated targets were Sall1, Foxa2, Nf2, Ctnnb1, Shh, and Bmpr1a. Therefore, these data suggested that TFs can drive the reprogramming of somatic cells towards a pluripotent state via neuroectoderm. Moreover, the neural lineage reprogramming system can address how miRNAs influence their target sites.
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Abbreviations
- iPSCs:
-
Induced pluripotent stem cells
- Pdx1:
-
Pancreatic and duodenal homeobox 1
- Ascl1:
-
Achaete-scute family BHLH transcription factor 1
- PCR:
-
Polymerase chain reaction
- Sox1:
-
Sex determining region Y-box 1
- PSA-NCAM:
-
Polysialylated neural cell adhesion molecule
- AKP:
-
Alkaline phosphatase
- LIF:
-
Leukemia inhibitory factor
- GFAP:
-
Glial fibrillary acidic protein
- α-SMA:
-
Alpha-smooth muscle aorta
- Afp:
-
Alpha-fetoprotein
- dpc:
-
Day post coitus
- ICM:
-
Inner cell mass
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Funding
The design of the study and the collection of the data were supported by the National Nature Science Foundation of China (Nos. 31601171, 31000645). The analysis of the data was supported by Heilongjiang Postdoctoral Science Foundation (No. 314000284) and Natural Science Foundation of Heilongjiang Province (No. C2018040). The interpretation of data and the writing of the manuscript were supported by the Fundamental Research Funds for the Provincial Universities (No. UNPYSCT-2018055), and Yu Weihan Foundation of Distinguished Young Scholars.
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Animal experiments were performed in accordance with the University of Health Guide for the Care and Use of Laboratory Animals and were approved under the code of Practice Harbin Medicine University Ethics Committees (HMUIRB20090012).
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10735_2022_10082_MOESM3_ESM.xlsx
Table S3. The predicted miRNA target mRNAs were identified from 4TF cells and Con cells on D3. Supplementary file3 (XLSX 157 kb)
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Sun, R., Gong, T., Liu, H. et al. Identification of microRNAs related with neural germ layer lineage-specific progenitors during reprogramming. J Mol Histol 53, 623–634 (2022). https://doi.org/10.1007/s10735-022-10082-w
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DOI: https://doi.org/10.1007/s10735-022-10082-w