Abstract
Although the human brain would be an ideal model for studying human neuropathology, it is difficult to perform in vitro culture of human brain cells from genetically engineered healthy or diseased brain tissue. Therefore, a suitable model for studying the molecular mechanisms responsible for neurological diseases that can appropriately mimic the human brain is needed. Somatic cell nuclear transfer (SCNT) was performed using an established porcine Yucatan EGFP cell line and whole seeding was performed using SCNT blastocysts. Two Yucatan EGFP porcine embryonic stem-like cell (pESLC) lines were established. These pESLC lines were then used to establish an in vitro neuro-organoids. Aggregates were cultured in vitro until 61 or 102 days after neural induction, neural patterning, and neural expansion. The neuro-organoids were sampled at each step and the expression of the dopaminergic neuronal marker (TH) and mature neuronal marker (MAP2) was confirmed by reverse transcription-PCR. Expression of the neural stem cell marker (PAX6), neural precursor markers (S100 and SOX2), and early neural markers (MAP2 and Nestin) were confirmed by immunofluorescence staining. In conclusion, we successfully established neuro-organoids derived from pESLCs in vitro. This protocol can be used as a tool to develop in vitro models for drug development, patient-specific chemotherapy, and human central nervous system disease studies.
Highlights
Studies of human neuropathologies are hindered by limitations of in vitro culture of the human brain for genetically engineering healthy or diseased brain tissue
Structural damage to these mitochondrial DNA (mtDNA) genes can reduce the energy required for differentiated cells, thereby limiting their therapeutic potential and their affecting their application in disease modeling orscreening
embryoid body (EB) formation was induced to confirm the ability of the established cell lines to differentiate into the three germ layers of the established porcine embryonic stemlike cell (pESLC) lines
Summary
Studies of human neuropathologies are hindered by limitations of in vitro culture of the human brain for genetically engineering healthy or diseased brain tissue. Qian et al [8] developed human iPSCs for 7 days to form patterned EBs with region-specific patterning factors, which were coated on Matrigel for 7 days and mass-cultured in a small rotating bioreactor This approach improved organoid culture reproducibility and successfully produced other brain areaspecific organs, including the whole brain, midbrain, and hypothalamic organisms [8]. Sci. 2021, 22, 2600 damage was increased in iPSCs established using cell lines from elderly patients compared to those derived from younger patients Structural damage to these mtDNA genes can reduce the energy required for differentiated cells, thereby limiting their therapeutic potential and their affecting their application in disease modeling orscreening.
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