Exploring the molecular mechanisms of MSC-derived exosomes in Alzheimer's disease: Autophagy, insulin and the PI3K/Akt/mTOR signaling pathway.

Alzheimer's disease (AD) is a devastating neurological condition characterized by cognitive decline, motor coordination impairment, and amyloid plaque accumulation. The underlying molecular mechanisms involve oxidative stress, inflammation, and neuronal degeneration. This study aimed to investigate the therapeutic effects of mesenchymal stem cell-derived exosomes (MSC-exos) on AD and explore the molecular pathways involved, including the PI3K/Akt/mTOR axis, autophagy, and neuroinflammation. To assess the potential of MSC-exos for the treatment of AD, rats were treated with AlCl3 (17 mg/kg/once/day) for 8 weeks, followed by the administration of an autophagy activator (rapamycin), or MSC-exos with or without an autophagy inhibitor (3-methyladenin; 3-MA+ chloroquine) for 4 weeks. Memory impairment was tested, and brain tissues were collected for gene expression analyses, western blotting, histological studies, immunohistochemistry, and transmission electron microscopy. Remarkably, the administration of MSC-exos improved memory performance in AD rats and reduced the accumulation of amyloid-beta (Aß) plaques and tau phosphorylation. Furthermore, MSC-exos promoted neurogenesis, enhanced synaptic function, and mitigated astrogliosis in AD brain tissues. These beneficial effects were associated with the modulation of autophagy and the PI3K/Akt/mTOR signalling pathway, as well as the inhibition of neuroinflammation. Additionally, MSC-exos were found to regulate specific microRNAs, including miRNA-21, miRNA-155, miRNA-17-5p, and miRNA-126-3p, further supporting their therapeutic potential. Histopathological and bioinformatic analyses confirmed these findings. This study provides compelling evidence that MSC-exos hold promise as a potential therapeutic approach for AD. By modulating the PI3K/Akt/mTOR axis, autophagy, and neuroinflammation, MSC-exos have the potential to improve memory, reduce Aß accumulation, enhance neurogenesis, and mitigate astrogliosis. These findings shed light on the therapeutic potential of MSC-exos and highlight their role in combating AD.

3D in-vitro cultures of human bone marrow and Wharton's jelly derived mesenchymal stromal cells show high chondrogenic potential.

In this study, chondrogenic potentials of 3D high-density cultures of Bone Marrow (BM) and Wharton's Jelly (WJ)-derived mesenchymal stromal cells (MSCs) was investigated by chondrogenesis- and cytokine-related gene expression over a 16-day culture period supplemented with human transforming growth factor (hTGF)-ß1 at 10 ng/ml. In BM-MSC 3D models, a marked upregulation of chondrogenesis-related genes, such as SOX9, COL2A1, and ACAN (all p < 0.05) and formation of spherical pellets with structured type II collagen fibers were observed. Similarly, WJ-based high-density culture appeared higher in size and more regular in shape, with a significant overexpression of COL2A1 and ACAN (all p < 0.05) at day 16. Moreover, a similar upregulation trend was documented for IL-6 and IL-10 expression in both BM and WJ 3D systems. In conclusion, MSC-based high-density cultures can be considered a promising in vitro model of cartilage regeneration and tissue engineering. Moreover, our data support the use of WJ-MSCs as a valid alternative for chondrogenic commitment of stem cells in regenerative medicine.

Effect of bone marrow mesenchymal stem cells-derived exosomes on diabetes-induced retinal injury: Implication of Wnt/ b-catenin signaling pathway.

BACKGROUND: Diabetic retinopathy (DR) is a serious microvascular complication of diabetes mellitus. Mesenchymal stem cells are currently studied as therapeutic strategy for management of DR. Exosomes, considered as a promising cell-free therapy option, display biological functions similar to those of their parent cells. In retinal development, Wnt/b-catenin signaling provides key cues for functional progression. The present study aimed to evaluate the potential efficacy of bone marrow-derived mesenchymal stem cell-derived exosomes (BM-MSCs-Ex) in diabetes-induced retinal injury via modulation of the Wnt/ b-catenin signaling pathway. METHODS: Eighty-one rats were allocated into 6 groups (control, DR, DR + DKK1, DR + exosomes, DR + Wnt3a and DR + exosomes+Wnt3a). Evaluation of each group was via histopathological examination, assessment of gene and/or protein expression concerned with oxidative stress (SOD1, SOD2, Nox2, Nox4, iNOS), inflammation (TNF-α, ICAM-1, NF-κB) and angiogenesis (VEGF, VE-cadherin). RESULTS: Results demonstrated that exosomes blocked the wnt/b-catenin pathway in diabetic retina concomitant with significant reduction of features of DR as shown by downregulation of retinal oxidants, upregulation of antioxidant enzymes, suppression of retinal inflammatory and angiogenic markers. These results were further confirmed by histopathological results, fundus examination and optical coherence tomography. Additionally, exosomes ameliorative effects abrogated wnt3a-triggered retinal injury in DR. CONCLUSION: Collectively, these data demonstrated that exosomes ameliorated diabetes-induced retinal injury via suppressing Wnt/ b-catenin signaling with subsequent reduction of oxidative stress, inflammation and angiogenesis.

Dimethyloxalylglycine (DMOG), a Hypoxia Mimetic Agent, Does Not Replicate a Rat Pheochromocytoma (PC12) Cell Biological Response to Reduced Oxygen Culture.

Cells respond to reduced oxygen availability predominately by activation of the hypoxia-inducible factor (HIF) pathway. HIF activation upregulates hundreds of genes that help cells survive in the reduced oxygen environment. The aim of this study is to determine whether chemical-induced HIF accumulation mimics all aspects of the hypoxic response of cells. We compared the effects of dimethyloxalylglycine (DMOG) (a HIF stabiliser) on PC12 cells cultured in air oxygen (20.9% O2, AO) with those cultured in either intermittent 20.9% O2 to 2% O2 (IH) or constant 2% O2 (CN). Cell viability, cell cycle, HIF accumulation, reactive oxygen species (ROS) formation, mitochondrial function and differentiation were used to characterise the PC12 cells and evaluate the impact of DMOG. IH and CN culture reduced the increase in cell numbers after 72 and 96 h and MTT activity after 48 h compared to AO culture. Further, DMOG supplementation in AO induced a dose-dependent reduction in the increase in PC12 cell numbers and MTT activity. IH-cultured PC12 cells displayed increased and sustained HIF-1 expression over 96 h. This was accompanied by increased ROS and mitochondrial burden. PC12 cells in CN displayed little changes in HIF-1 expression or ROS levels. DMOG (0.1 mM) supplementation resulted in an IH-like HIF-1 profile. The mitochondrial burden and action potential of DMOG-supplemented PC12 cells did not mirror those seen in other conditions. DMOG significantly increased S phase cell populations after 72 and 96 h. No significant effect on PC12 cell differentiation was noted with IH and CN culture without induction by nerve growth factor (NGF), while DMOG significantly increased PC12 cell differentiation with and without NGF. In conclusion, DMOG and reduced oxygen levels stabilise HIF and affect mitochondrial activity and cell behaviour. However, DMOG does not provide an accurate replication of the reduced oxygen environments.

Functional Recellularization of Acellular Rat Liver Scaffold by Induced Pluripotent Stem Cells: Molecular Evidence for Wnt/B-Catenin Upregulation.

BACKGROUND: Liver transplantation remains the only viable therapy for liver failure but has a severely restricted utility. Here, we aimed to decellularize rat livers to form acellular 3D bio-scaffolds suitable for seeding with induced pluripotent cells (iPSCs) as a tool to investigate the role of Wnt/ß-catenin signaling in liver development and generation. METHODS: Dissected rat livers were randomly divided into three groups: I (control); II (decellularized scaffolds) and III (recellularized scaffolds). Liver decellularization was established via an adapted perfusion procedure and assessed through the measurement of extracellular matrix (ECM) proteins and DNA content. Liver recellularization was assessed through histological examination and measurement of transcript levels of Wnt/ß-catenin pathway, hepatogenesis, liver-specific microRNAs and growth factors essential for liver development. Adult rat liver decellularization was confirmed by the maintenance of ECM proteins and persistence of growth factors essential for liver regeneration. RESULTS: iPSCs seeded rat decellularized livers displayed upregulated transcript expression of Wnt/ß-catenin pathway-related, growth factors, and liver specification genes. Further, recellularized livers displayed restored liver-specific functions including albumin secretion and urea synthesis. CONCLUSION: This establishes proof-of-principle for the generation of three-dimensional liver organ scaffolds as grafts and functional re-establishment.

3D Biomimetic Scaffold for Growth Factor Controlled Delivery: An In-Vitro Study of Tenogenic Events on Wharton's Jelly Mesenchymal Stem Cells.

The present work described a bio-functionalized 3D fibrous construct, as an interactive teno-inductive graft model to study tenogenic potential events of human mesenchymal stem cells collected from Wharton's Jelly (hWJ-MSCs). The 3D-biomimetic and bioresorbable scaffold was functionalized with nanocarriers for the local controlled delivery of a teno-inductive factor, i.e., the human Growth Differentiation factor 5 (hGDF-5). Significant results in terms of gene expression were obtained. Namely, the up-regulation of Scleraxis (350-fold, p ≤ 0.05), type I Collagen (8-fold), Decorin (2.5-fold), and Tenascin-C (1.3-fold) was detected at day 14; on the other hand, when hGDF-5 was supplemented in the external medium only (in absence of nanocarriers), a limited effect on gene expression was evident. Teno-inductive environment also induced pro-inflammatory, (IL-6 (1.6-fold), TNF (45-fold, p ≤ 0.001), and IL-12A (1.4-fold)), and anti-inflammatory (IL-10 (120-fold) and TGF-ß1 (1.8-fold)) cytokine expression upregulation at day 14. The presented 3D construct opens perspectives for the study of drug controlled delivery devices to promote teno-regenerative events.

Dose-Response Tendon-Specific Markers Induction by Growth Differentiation Factor-5 in Human Bone Marrow and Umbilical Cord Mesenchymal Stem Cells.

Mesenchymal stem cells derived from human bone marrow (hBM-MSCs) are utilized in tendon tissue-engineering protocols while extra-embryonic cord-derived, including from Wharton's Jelly (hWJ-MSCs), are emerging as useful alternatives. To explore the tenogenic responsiveness of hBM-MSCs and hWJ-MSCs to human Growth Differentiation Factor 5 (hGDF-5) we supplemented each at doses of 1, 10, and 100 ng/mL of hGDF-5 and determined proliferation, morphology and time-dependent expression of tenogenic markers. We evaluated the expression of collagen types 1 (COL1A1) and 3 (COL3A1), Decorin (DCN), Scleraxis-A (SCX-A), Tenascin-C (TNC) and Tenomodulin (TNMD) noting the earliest and largest increase with 100 ng/mL. With 100 ng/mL, hBM-MSCs showed up-regulation of SCX-A (1.7-fold) at Day 1, TNC (1.3-fold) and TNMD (12-fold) at Day 8. hWJ-MSCs, at the same dose, showed up-regulation of COL1A1 (3-fold), DCN (2.7-fold), SCX-A (3.8-fold) and TNC (2.3-fold) after three days of culture. hWJ-MSCs also showed larger proliferation rate and marked aggregation into a tubular-shaped system at Day 7 (with 100 ng/mL of hGDF-5). Simultaneous to this, we explored the expression of pro-inflammatory (IL-6, TNF, IL-12A, IL-1ß) and anti-inflammatory (IL-10, TGF-ß1) cytokines across for both cell types. hBM-MSCs exhibited a better balance of pro-inflammatory and anti-inflammatory cytokines up-regulating IL-1ß (11-fold) and IL-10 (10-fold) at Day 8; hWJ-MSCs, had a slight expression of IL-12A (1.5-fold), but a greater up-regulation of IL-10 (2.5-fold). Type 1 collagen and tenomodulin proteins, detected by immunofluorescence, confirming the greater protein expression when 100 ng/mL were supplemented. In the same conditions, both cell types showed specific alignment and shape modification with a length/width ratio increase, suggesting their response in activating tenogenic commitment events, and they both potential use in 3D in vitro tissue-engineering protocols.

BMP2 repression and optimized culture conditions promote human bone marrow-derived mesenchymal stem cell isolation.

AIM: Human mesenchymal stem cells (hMSC) are multipotent progenitor cells. We propose the optimization of hMSC isolation and recovery using the application of a controlled hypoxic environment. MATERIALS & METHODS: We evaluated oxygen, glucose and serum in the recovery of hMSC from bone marrow (BMhMSC). Colony forming units-fibroblastic, cell numbers, tri-lineage differentiation, immunofluorescence and microarray were used to confirm and characterize BMhMSC. RESULTS: In an optimized (2% O(2), 4.5 g/l glucose and 5% serum) environment both colony forming units-fibroblastic (p = 0.01) and cell numbers (p = 0.0001) were enhanced over standard conditions. Transcriptional analysis identified differential expression of bone morphogenetic protein 2 (BMP2) and, putatively, chemokine (C-X-C motif) receptor 2 (CXCR2) signaling pathways. CONCLUSION: We have detailed a potential milestone in the process of refinement of the BMhMSC isolation process.

Vibrational spectroscopy differentiates between multipotent and pluripotent stem cells.

Over the last few years, there has been an increased interest in the study of stem cells in biomedicine for therapeutic use and as a source for healing diseased or injured organs/tissues. More recently, vibrational spectroscopy has been applied to study stem cell differentiation. In this study, we have used both synchrotron based FTIR and Raman microspectroscopies to assess possible differences between human pluripotent (embryonic) and multipotent (adult mesenchymal) stem cells, and how O(2) concentration in cell culture could affect the spectral signatures of these cells. Our work shows that infrared spectroscopy of embryonic (pluripotent) and adult mesenchymal (multipotent) stem cells have different spectral signatures based on the amount of lipids in their cytoplasm (confirmed with cytological staining). Furthermore, O(2) concentration in cell culture causes changes in both the FTIR and Raman spectra of embryonic stem cells. These results show that embryonic stem cells might be more sensitive to O(2) concentration when compared to mesenchymal stem cells. While vibrational spectroscopy could therefore be of potential use in identifying different populations of stem cells further work is required to better understand these differences.