Studies have shown that stem cells from different sources have the ability to form neurons and glia after transplantation, and help the regeneration of surviving sensory neurons, especially in the DRG cavity and the damaged area of DRG. Sacai et alshowed that neuroectodermal progenitor cells in human bone marrow stromal cells (hBMSCs) can be selectively expanded and then induced to differentiate into Schwann like cells. The co-culture of Schwann like cells and embryonic DRG neurons promotes the formation of Schwann like cells, which has been proved to be related to injury repair and myelination in vivo. There are a large number of high-purity human neuronal progenitors (hNPs) in hESCs, and when hESCs are transplanted into the injured spinal cord, hNPs are naturally integrated into the host tissue and various mature neuronal subtypes are generated, and the whole process is deeply affected by the cellular microenvironment. Kitazawa A's teamfound that the combination of medium (CM) and nerve growth factor (NGF) in chicken back root ganglion (DRG) conditions had an impact on the directional differentiation of ESCs. They confirmed that DRG-CM can effectively promote ESCs differentiation into neurons. Vidal Midentified neural crest-derived stem cells in DRG; in vitro, these cells are able to form pluripotent spheroids that produce neurons, glial cells, and myofibroblasts, and while DRG appears to have stem cell potential, their identity and pathophysiology remain unknown. In order to confirm whether DRG itself or after induction by stem cells has stem cell properties, researchers are still exploring.
In this study, 4102 genes with different expression between undifferentiated hESCs and DRG differentiated by hESCs for one day were screened, including 2674 up-regulated genes and 1428 down regulated genes. Go analysis showed that the enriched genes were mainly involved in biological regulation, metabolic process, cell membrane and nuclear synthesis, intimal system, regulating protein, regulating nucleic acid and other metabolic processes. KEGG analysis showed that the differential genes were mainly distributed in axon guidance, signaling pathways regulating pluripotency of stem cells, pathways in cancer, ECM receiver interaction, focal adhesion, cell adhesion molecules (CAMs), PI3K Akt signaling pathway, MAPK signaling pathway, TGF beta signaling pathway and Ras signaling pathway; In the signal pathway regulating stem cell pluripotency, 34 genes were up-regulated and 13 genes were down-regulated. Finally, differential genes analyze the signal pathway regulating stem cell pluripotency through PPI network. It finds that the core protein interaction in the up-regulated genes is stable. Based on the analysis of Cytoscape software algorithm, the common hub genes on this pathway are BMP4, SOX2, FGF2, Nanog, Smad3, KLF4, GS3B and FGFR2.
In the above hub gene, the up-regulated genes include BMP4, SOX2, FGF2, NANOG, SMAD3, KLF4, FGFR2, and the downregulated gene is GSK3B. Bone morphogenetic protein 4 (BMP4) induces hESCs to differentiate into a trophoblast layer. This induction process of BMP4 is mainly achieved by inhibiting the extracellular receptor kinase (ERK) and p38 mitosogen-activated protein kinase (MAPK) pathways. In DRG, BMP4 is able to inhibit synaptic growth. SOX2 is a key regulator of a variety of stem cells, particularly ESCs and neural progenitor cells (NPCs). Understanding the functional mechanisms of SOX2 can help realize the potential of ESC and NPCs. SOX2 inhibits non-neural lineages in hESCs and regulates neurogenesis in hNPC by inhibiting classical Wnt signaling. Cells in DRG capable of highly expressing neural crest cell markers include Nestin, Sox2, Sox10, and p75, where Sox2-positive cells, especially satellite glial cells associated with chronic pain, can induce neurogenesis[19, 20]. Fibroblast growth factor 2 (FGF2) promotes self-renewal of hESCs. hESCs that overexpress FGF2 have a neuroprotective effect and can shift the body to an anti-inflammatory environment. However, FGF2 inhibits the synaptic growth of dorsal root ganglion neurons (DRGN). NANOG is a key transcription factor for ESCs pluripotency. NANOG has three paralogous homologs in human cells, namely NANOG1, NANOG2 and NANOGP8, hESCs express large amounts of NANOG1 and NANO2. NanoGP8, NANOGP4 and NANOGP5 are expressed in human cancer cells. Notably, in some cancer cells, NANOGP8 produces levels of the NANOG protein comparable to those produced by NANOG1 in pluripotent cells; cancer-associated NANOGP8 helps promote dedifferentiation and cell plasticity. The transcriptional regulation of NANOG itself in hESCs is largely elusive, and Chan's findings show that NANOG 's two novel upstream transcriptional activators are functionally important for self-renewal of hESCs. However, NANOG coverage in DRG is almost non-existent. SMAD3 is an intracellular medium that transduces signals from transforming growth factor (TGF) and activin receptors. Efficient transformation of hESCs and iPS cells in the nervous system can be achieved by inhibiting SMAD signaling. The expression of the activator downstream signaling molecule, SMAD3 mRNA, decreases with the development of DRG in chicken embryos. Elisa results showed that activin A through SMAD3 may play an important role in the early development of embryonic DRG, which is associated with inhibiting glial cell proliferation and regulating the release of the neurotransmitter GABA. Krüppel-like factor 4 (KLF4) directly regulates hESCs, Gaining insight into the regulation of core transcription factors helps to better control the self-renewal and pluripotency of hESCs. However, the transcriptional regulation of NANOG itself in hESCs is largely elusive. Related studies have found that during the differentiation of hESCs, the mRNA and protein expressions of KLF4 and PBX1 are down-regulated. In addition, the overexpression of KLF4 and PBX1 upregulated the activity of THE NANOGO promoter in hESCs and the expression of endogenous NANOG proteins, thus affecting the self-renewal of hESCs. Although several components of the fibroblast growth factor (FGF) signaling pathway have been reported to be detected in hESCs, the function of this pathway and its effect on cell fate decisions have not been determined. In Petr's study, the expression of FGF-2 and its receptors (FGFRs) in undifferentiated and differentiated hESCs was found. FGF-2 can influence hESCs as exogenous and endogenous factors. In addition, hESCs release FGF-2 into the medium, which indicates that they have autocrine activity. FGFR activation in DRG was found after peripheral nerve injury (PNSI) in rats, but this activation process is mostly associated with pain. Glycogen synthase kinase 3 (GSK3 ) is a key enzyme involved in glycogen metabolism, but is now known to regulate a variety of cellular functions. In ESCs culture, certain small molecules alter key signaling pathways to promote cell self-renewal and inhibit differentiation. In mice, for example, small molecules in the FGF/MEK/Erk and GSK3B pathways inhibit the formation of the mesoderm. GSK-3B inhibitor TDZD-8 has a certain effect on the axon growth of dorsal rhizobia neurons in neonatal rats, low concentration of TDZD-8 can promote axonal growth, the formation of multiple axons or axon branches, while high concentration of TDZD-8 significantly inhibits axonal growth, resulting in axonal retraction. In general, reports of hub gene-related genes regulating the signaling pathways of stem cell pluripotency are more common in hESCs, most of which are transcription factors, which have clearly dominated the self-renewal and cell fate of ESCs; although transcriptional regulators have been extensively studied in hESCs, the extent of their contribution to pluripotency is still unclear.
Bioinformatics technology was used to analyze gene chip data of undifferentiated hESCs and DRG that induced hESCs differentiation for 1 day, and the GEO2R analysis tool, webgestalt database, STRING analysis and Cytoscape software were used to screen out the differentially expressed genes in undifferentiated hESCs and DRG that induced hESCs differentiation for 1 day, and conducted in-depth research on the relationship between biological function and protein interaction network. To explore the possible molecular mechanisms of differential genes expressed, it will help to study the clinical treatment of stem cells.
In summary, the results of this study show that there is a significant expression difference gene between undifferentiated hESCs and DRG that induces the differentiation of hESCs for 1 day, and signaling pathways that regulate stem cell pluripotency are analyzed, and it is found that BMP4, SOX2, FGF2, NANOG, SMAD3, KLF4, GSK3B, FGFR2 are closely related to stem cell self-renewal in undifferentiated hESCs. In DRG, where hESCs differentiate for 1 day, BMP4, SOX2, FGF2, SMAD3, GSK3B, and FGFR2 are mostly related to neuropathic pain and neurite growth, and their stem cell characteristics need to be further explored.