Isolation of human mesenchymal stem cells from the skin and their neurogenic differentiationin vitro

Abstract

(J Korean Assoc Oral Maxillofac Surg 2012;38:343-53) Objectives: This aim of this study was to effectively isolate mesenchymal stem cells (hSMSCs) from human submandibular skin tissues (termed hSMSCs) and evaluate their characteristics. These hSMSCs were then chemically induced to the neuronal lineage and analyzed for their neurogenic characteristics in vitro. Materials and Methods: Submandibular skin tissues were harvested from four adult patients and cultured in stem cell media. Isolated hSMSCs were evaluated for their multipotency and other stem cell characteristics. These cells were differentiated into neuronal cells with a chemical induction protocol. During the neuronal induction of hSMSCs, morphological changes and the expression of neuron-specific proteins (by fluorescence-activated cell sorting [FACS]) were evaluated. Results: The hSMSCs showed plate-adherence, fibroblast-like growth, expression of the stem-cell transcription factors Oct 4 and Nanog, and positive staining for mesenchymal stem cell (MSC) marker proteins (CD29, CD44, CD90, CD105, and vimentin) and a neural precursor marker (nestin). Moreover, the hSMSCs in this study were successfully differentiated into multiple mesenchymal lineages, including osteocytes, adipocytes, and chondrocytes. Neuron-like cell morphology and various neural markers were highly visible six hours after the neuronal induction of hSMSCs, but their neuron-like characteristics disappeared over time (24-48 hrs). Interestingly, when the chemical induction medium was changed to Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS), the differentiated cells returned to their hSMSC morphology, and their cell number increased. These results indicate that chemically induced neuron-like cells should not be considered true nerve cells. Conclusion: Isolated hSMSCs have MSC characteristics and express a neural precursor marker, suggesting that human skin is a source of stem cells. However, the in vitro chemical neuronal induction of hSMSC does not produce long-lasting nerve cells and more studies are required before their use in nerve-tissue transplants.

Cholinergic Nerve Differentiation of Mesenchymal Stem Cells Derived from Long-Term Cryopreserved Human Dental Pulp In Vitro and Analysis of Their Motor Nerve Regeneration Potential In Vivo

“…After cholinergic neuronal induction of hDPSCs-cryo, immunostaining for cholinergic neuron-specific marker proteins was performed using previously described protocols [ 37 ]. Briefly, DF-chN were fixed with 3.7% formaldehyde for 40 min, permeabilized with 0.2% Triton X-100 supplemented with 1% BSA, and thereafter blocked with 1% BSA (in DPBS) at 20 °C for 1 h. Cells were then incubated with primary antibody, diluted 1:100, choline acetyltransferase (ChAT; ab68779, Abcam, Cambridge, UK), homeobox HB9 (motor neuron and pancreas homeobox 1, MNX1) (HB9; sc-22542, Santa Cruz), and insulin gene enhancer protein ISL-1 (ISL1; sc101072, Santa Cruz) at 20 °C for 1 h. Following incubation with primary antibodies, cells were washed with DPBS and incubated with 1:200 CruzFluor TM 594 or CruzFluor TM 488 conjugated donkey anti-rabbit or donkey anti-goat or donkey anti-mouse IgG secondary antibodies (Santa Cruz) for 1 h. For nuclear staining, cells were treated with 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at 20 °C.…”

Using Stem Cells to Grow Artificial Tissue for Peripheral Nerve Repair

“…Once the inner cell mass is disassociated from the embryo, the originator is obliged to donate samples to the United Kingdom Stem Cell Bank (UKSCB) and the stem cell line is regulated by the Human Tissue Authority [ 130 ]. Isolation of mesenchymal stem cells can involve multistage ex vivo processing and numerous protocols are available [ 131 – 135 ].…”

Neuronal transcription program induced in hippocampal cells cocultured with bone marrow derived mesenchymal cells

“…This sustained neuronal morphology might be due to true differentiation of BMSCs with the help of hippocampal cells as previous reports suggests that the cocultured of neuronal progenitor cells (NPCs) and BMSCs stimulates BMSCs to differentiate into neural stem cells and neurons [ 34 ]. In other protocols neuronal like morphology returns toward original cell morphology as chemical treatment discontinued [ 35 ]. After proliferation and morphological evaluation, we next examined the expression of neural progenitor markers nestin and tubulin for identification of immature neurons in all groups.…”