Conditioned medium-enriched umbilical cord mesenchymal stem cells: a potential therapeutic strategy for spinal cord injury, unveiling transcriptomic and secretomic insights.

INTRODUCTION: Spinal cord injury (SCI) leads to significant destruction of nerve tissue, causing the degeneration of axons and the formation of cystic cavities. This study aimed to examine the characteristics of human umbilical cord-derived mesenchymal stem cells (HUCMSCs) cultured in a serum-free conditioned medium (CM) and assess their effectiveness in a well-established hemitransection SCI model. MATERIALS AND METHODS: In this study, HUCMSCs cultured medium was collected and characterized by measuring IL-10 and identifying proteomics using mass spectroscopy. This collected serum-free CM was further used in the experiments to culture and characterize the HUMSCs. Later, neuronal cells derived from CM-enriched HUCMSC were tested sequentially using an injectable caffeic acid-bioconjugated gelatin (CBG), which was further transplanted in a hemitransection SCI model. In vitro, characterization of CM-enriched HUCMSCs and differentiated neuronal cells was performed using flow cytometry, immunofluorescence, electron microscopy, and post-transplant analysis using immunohistology analysis, qPCR, in vivo bioluminescence imaging, and behavioral analysis using an infrared actimeter. RESULTS: The cells that were cultured in the conditioned media produced a pro-inflammatory cytokine called IL-10. Upon examining the secretome of the conditioned media, the Kruppel-like family of KRAB and zinc-finger proteins (C2H2 and C4) were found to be activated. Transcriptome analysis also revealed an increased expression of ELK-1, HOXD8, OTX2, YY1, STAT1, ETV7, and PATZ1 in the conditioned media. Furthermore, the expression of Human Stem-101 confirmed proliferation during the first 3 weeks after transplantation, along with the migration of CBG-UCNSC cells within the transplanted area. The gene analysis showed increased expression of Nestin, NeuN, Calb-2, Msi1, and Msi2. The group that received CBG-UCNSC therapy showed a smooth recovery by the end of week 2, with most rats regaining their walking abilities similar to those before the spinal cord injury by week 5. CONCLUSIONS: In conclusion, the CBG-UCNSC method effectively preserved the integrity of the transplanted neuronal-like cells and improved locomotor function. Thus, CM-enriched cells can potentially reduce biosafety risks associated with animal content, making them a promising option for clinical applications in treating spinal cord injuries.

Human gingival derived neuronal cells in the optimized caffeic acid hydrogel for hemitransection spinal cord injury model.

Spinal cord injury induces scar formation causes axonal damage that leads to the degeneration of axonal function. Still, there is no robust conceptual design to regenerate the damaged axon after spinal injury. Therefore, the present study demonstrates that human gingival derived neuronal stem cells (GNSCs) transplants in the injectable caffeic acid bioconjugated hydrogel (CBGH) helps to bridge the cavity and promote the engraftment and repopulation of transplants in the injured spinal tissue. Our study reports that the bioluminescence imaging in vivo imaging system (IVIS) provides a satisfactory progression in CBGH-GNSCs transplants compare to lesion control and CBGH alone. Immune regulators interleukin-6 (IL-6), tumor necrosis factor-α, neutrophil elastase are decreased, IL-10 is increased. Likewise, immunostaining (TAU/TUJ-1, SOX-2/NeuN, MAP-2/PSD93, NSE, S100b, and GFAP) shown repopulated cells. Also, TRA-1-81 expression confirms the absence of immune rejection in the CBGH-GNSCs transplants. However, locomotor recovery test, gene (IL-6, CASPASE3, p14-ARF, VEGF, LCAM, BDNF, NT3, NGN2, TrKc, FGF2, Sox-2, TUJ-1, MAP-2, Nestin, and NeuN) and protein expression (TAU, TUJ-1, SOX-2 MAP-2, PSD93, NeuN, TRA-1-81, GFAP, TAU, and MBP) shows functional improvements in the CBGH-GNSCs group. Further, GABA and glutamine level demonstrates the new synaptic vesicle formation. Hence, the CBGH scaffold enhances GNSCs transplants to restore the injured spinal tissue.

Passage-dependent expression of STRO-1 in human gingival mesenchymal stem cells.

The expression of STRO-1, the essential mesenchymal stem cell marker, was found to decrease with advancing passages in few tissues. Because STRO-1 was identified and isolated from human gingiva, we were interested to know its status after a few passages. Human gingival mesenchymal stem cells (HGMSCs) were isolated from human gingiva. Flow cytometry was carried out with STRO-1, mesenchymal stem cell (MSC) positive marker CD73, and negative marker CD34/CD45. Samples were also subjected to CD90 and STRO-1 immunofluorescence staining. Gene expression was carried out for transcription factors OCT-4, NANOG, and NESTIN. The results showed a gradual decrease in STRO-1 and transcription factor expression with an increase in passage numbers. MSC positive marker CD73 was consistently expressed in all the passages. Negative markers were absent in all the passages. We conclude that STRO-1 may be a useful marker to isolate undifferentiated (potent) mesenchymal cells from gingiva.

Transdifferentiation of human gingival mesenchymal stem cells into functional keratinocytes by Acalypha indica in three-dimensional microenvironment.

Gingival tissue is reportedly a promising, easily accessible, abundant resource of mesenchymal stem cells (MSC) for use in various tissue engineering strategies. Human gingival MSC (HGMSCs) were successfully isolated from gingival tissue and characterized. To analyze in a two-dimensional form, HGMSCs were cultured with basal medium and induced with 25 µg/ml of Acalypha indica. Quantitative real-time polymerase chain reaction (qPCR) and western blot analysis showed the presence of keratinocyte-specific markers, including cytokeratin-5 and involucrin. To further assess its capability for stratification akin to human keratinocytes, HGMSCs were encapsulated in a HyStem® -HP Cell Culture Scaffold Kit and cultured in the presence of A. indica. Calcein AM staining indicated that the HyStem® -HP Scaffold Kit has excellent biocompatibility. Immunofluorescence and qPCR analysis revealed the presence of keratinocyte-specific markers. The study concluded that the three-dimensional microenvironment is a novel method for inducing epidermal differentiation of HGMSCs to engineer epidermal substitutes with the help of A. indica, which provides an alternative strategy for skin tissue engineering.

Differential expression of microRNAs let-7a, miR-125b, miR-100, and miR-21 and interaction with NF-kB pathway genes in periodontitis pathogenesis.

Periodontitis is a chronic inflammatory disease which is caused by destruction of the tissues that surrounds and supports the tooth. Deregulation of microRNAs has been reported to cause several inflammatory diseases such as autoimmune disease, chronic periodontitis, and cancer. In the present study, we have investigated the expression pattern of microRNAs let-7a, miR-125b, miR-100, miR-21, and RNA-binding protein LIN-28A among healthy individuals and chronic periodontitis patients. Total RNA was isolated from gingival tissue samples collected from 100 healthy individuals and 100 chronic periodontitis patients. The expression of microRNAs and LIN-28 was performed by qPCR. Target prediction for the microRNAs was done using miRWalk and miRTarbase online databases and the experimentally validated targets were analyzed for their molecular function, biological processes, and related pathways using gProfiler software. The expression analysis revealed that let-7a and miR-21 were upregulated, whereas, miR-100, miR-125b, and LIN-28 were down regulated. The age dependent expression analysis revealed that the expression levels of all the microRNAs and LIN-28 were found to increase with age (more than 50 years), thereby suggesting an increased risk to chronic periodontitis. Among the various targets predicted using miRWalk and miRTarbase databases, NFKB was found to be a common target among all the four microRNAs. gProfiler revealed several functions such as NF-ĸB signaling pathway, cytokine-cytokine receptor interaction, osteoclast differentiation, etc., all of which specific to inflammation and periodontitis.

Gingival spheroids possess multilineage differentiation potential.

Recently studies have demonstrated HGMSCs as ideal candidates for regenerative study. Interestingly we found that HGMSCs derived spheroids are more potent and maintain the properties of stemness convincingly compared to conventional culture methods. During the culture, GMSCs instinctively accumulated into spheroids and display multipotent STRO-1 and Vimentin-positive cells. Reduced phenotypic expression of CD73, CD105, and elevated expression STRO-1 and CD-34. Pluripotent nature of S-GMSCs putatively shown the expression of OCT4A, NANOG, SOX-2, SSEA4, TRA-1-60, and TRA-181. Also, levels of protein are much higher in spheroid than dissociated culture. On endothelial induction, spheroid differentiated and developed a vascular structure with positive expression of CD31 and on neuronal induction showed positivity for TUJ1 and E-Cadherin. Importantly, undifferentiated state of S-GMSCs exhibited significant upregulation of aforementioned pluripotent genes and lack of pro-inflammatory cytokines IL-6 and amplified ARF signal confirming that the spheroids are not teratoma formation. However, higher of CAP1, CP, TGFß, OPN, PPARÉ£, TUJ1, and NESTIN expression observed in spheroids, and minimal expression of the same markers were observed in adherent GMSCs respectively. Ahead of dissociated gingival culture, spheroid provides enhanced viable, pluripotent, and multilineage ability. This study suggested that S-GMSCs increased the chances of therapeutic efficacy in the regenerative applications.

Comparision of Gingival and Umbilical Cord Stem Cells Based on Its Modulus and Neuronal Differentiation.

The availability of Human Umbilical Cord-derived Mesenchymal Stem Cells (HUCMSCs) from a single sex being a major limitation for the utilization of a potential stem cell, it is highly desirable to utilize, an autogenous pluripotent cell with desirable biological and mechanical properties in clinical situations. Comparison of Human Gingival Mesenchymal Stem Cells (HGMSCs) with HUCMSCs demonstrates; MSCs derived from gingiva have higher proliferation rate and higher population doubling time than Umbilical Cord. Unlike HUCMSCs, immunofluorescence studies showed the presence of pluripotency markers OCT-4 and NANOG predominantly in the cytoplasm of HGMSCs which was confirmed by Western blot. The mechanical property, such as modulus of elasticity of HGMSCs, is on par with HUCMSCs, but the surface roughness found to be lesser in HGMSCs, which may suggest HGMSCs greater adhesive property to the extracellular matrix. There is a marginal difference in the neuronal differentiation rate between HGMSCs and HUCMSCs; both the cells expressed positivity for several neuronal lineage markers. Hence, HGMSCs represent an autogenous source of mesenchymal stem cells, which are easy to procure with least morbidity, multipotent in nature with desirable biological, and mechanical properties, probably an ideal candidate for clinical applications. J. Cell. Biochem. 118: 2000-2008, 2017. © 2017 Wiley Periodicals, Inc.

Osteogenic differentiation of human gingival mesenchymal stem cells by Aristolochia bracteolata supplementation through enhanced Runx2 expression.

The phenotypic characteristics of human gingival derived mesenchymal stem cells (HGMSCs) on induction with total methanol extract of Aristolochia bracteolata have been evaluated. HGMSCs were cultured in control and two different induction medium: Control medium (basal medium), OM1 (Standard induction medium), and OM2 (100 µg/ml of A. bracteolata). Osteogenic differentiation of the cultured cells was assessed by studying the calcium deposition and osteoblastic gene expression. OM2 medium showed an enhanced osteogenic differentiation potential than OM1 as measured by increased calcium deposition and elevated expression of Runx2, osteopontin, osteonectin, osteocalcin, Collagen type I, and ALP levels in comparison with OM1 differentiated cells. We conclude that at 100 µg/ml A. bracteolata has induced HGMSC differentiation into osteogenic lineage consequent to enhanced Runx2 expression and related osteogenic genes.