In Vitro Differentiation of Human Skin-Derived Cells into Functional Sensory Neurons-Like

Abstract

Skin-derived precursor cells (SKPs) are neural crest stem cells that persist in certain adult tissues, particularly in the skin. They can generate a large type of cell in vitro, including neurons. SKPs were induced to differentiate into sensory neurons (SNs) by molecules that were previously shown to be important for the generation of SNs: purmorphamine, CHIR99021, BMP4, GDNF, BDNF, and NGF. We showed that the differentiation of SKPs induced the upregulation of neurogenins. At the end of the differentiation protocol, transcriptional analysis was performed on BRN3A and a marker of pain-sensing nerve cell PRDM12 genes: 1000 times higher for PRDM12 and 2500 times higher for BRN3A in differentiated cells than they were in undifferentiated SKPs. Using immunostaining, we showed that 65% and 80% of cells expressed peripheral neuron markers BRN3A and PERIPHERIN, respectively. Furthermore, differentiated cells expressed TRPV1, PAR2, TRPA1, substance P, CGRP, HR1. Using calcium imaging, we observed that a proportion of cells responded to histamine, SLIGKV (a specific agonist of PAR2), polygodial (a specific agonist of TRPA1), and capsaicin (a specific agonist of TRPV1). In conclusion, SKPs are able to differentiate directly into functional SNs. These differentiated cells will be very useful for further in vitro studies.

A high-content platform for physiological profiling and unbiased classification of individual neurons

“…We did not observe a significant difference between edge and center wells in neuronal density (main effect of edge by univariate ANOVA, n = 316 edge wells per 543 center wells, p = 0.484); therefore, we continued to use all wells per plate. Traditional rig-based experiments generally use fewer than 100 cells per group ( Bataille et al, 2020 ; Baykara et al, 2019 ; Tonello et al, 2017 ); thus, using the lowest average cell count per well, we would only require two wells per group to surpass current typical sample sizes.…”

Pharmacological Manipulation of Wnt/β-Catenin Signaling Pathway in Human Neural Precursor Cells Alters Their Differentiation Potential and Neuronal Yield

“…Most studies using CHIR or XAV to guide differentiation of stem cells and other cell types used a final concentration of 2.5–10 μM ( De Kumar et al, 2017 ; Lancaster et al, 2017 ; Major et al, 2017 ; Shafa et al, 2018 ; Sharma et al, 2018 ; Srikanth et al, 2018 ; Yoon et al, 2019 ; Bataille et al, 2020 ). Yet, the same studies have not addressed whether these drugs have any cytotoxic effects on human or murine cell cultures.…”

“…Taking advantage of our in vitro drug screening platform of self-renewable hNPCs, we also asked whether CHIR or XAV might be cytotoxic, by measuring cell proliferation and survival. Both drugs were used in this study at the same concentration or lower as in similar studies employing human or murine ESCs, hiPSCs and other types of stem cells ( De Kumar et al, 2017 ; Malleske et al, 2018 ; Shafa et al, 2018 ; Gomez et al, 2019 ; Hamad et al, 2019 ; Qiu et al, 2019 ; Shin et al, 2019 ; Almasoud et al, 2020 ; Bataille et al, 2020 ; Bejoy et al, 2020 ; Govarthanan et al, 2020 ; Han et al, 2020 ; Kanagaki et al, 2020 ; Leigh et al, 2020 ; Yang J. et al, 2020 ; Yang Y. et al, 2020 ; Ren et al, 2021 ; Wang et al, 2021 ). When stem cells differentiate into more mature cell types, their proliferation potential is gradually reduced, until cells reach a post-mitotic fate.…”

“…Most relevant to the present report, is the use of CHIR and XAV as critical components of chemically defined protocols aiming to guide differentiation of human and murine stem cells into myriad cellular fates. CHIR has been used to promote neural and neuronal differentiation in embryonic stem cells and induced pluripotent stem cells ( De Kumar et al, 2017 ; Shafa et al, 2018 ; Gomez et al, 2019 ; Qiu et al, 2019 ; Shin et al, 2019 ; Bejoy et al, 2020 ) and in several different types of neural precursors ( Yang Y. et al, 2020 ; Ren et al, 2021 ; Wang et al, 2021 ), as well as to induce trans- differentiation into neural lineages from non-neural cell types, such as skin ( Bataille et al, 2020 ; Yang J. et al, 2020 ) and mesenchymal cells ( Govarthanan et al, 2020 ). XAV has been used to promote differentiation of pluripotent stem cells into lung cells ( Malleske et al, 2018 ; Kanagaki et al, 2020 ), to increase osteogenic differentiation of mesenchymal stem cells ( Almasoud et al, 2020 ; Han et al, 2020 ), and to promote generation of cardiomyocytes ( Hamad et al, 2019 ; Leigh et al, 2020 ).…”

A high-content platform for physiological profiling and unbiased classification of individual neurons

“…We did not observe a significant difference between edge and center wells in neuronal density (main effect of edge by univariate ANOVA, n=316 edge wells/543 center wells, F=0.49, p=0.484); therefore, we continued to use all wells per plate. Traditional rig-based experiments generally use fewer than 100 cells/group (Bataille et al, 2020;Baykara et al, 2019;Tonello et al, 2017); thus, using the lowest average cell count per well, we would only require two wells per group to surpass current typical sample sizes.…”