Nerve injuries are debilitating, leading to long-term motor deficits. Remyelination and axonal growth are supported and enhanced by growth factor and cytokines. Combination of nerve guidance conduits (NGCs) with adipose-tissue-derived multipotent mesenchymal stromal cells (AdMSCs) has been performing promising strategy for nerve regeneration. 3D-printed polycaprolactone (PCL)-NGCs were fabricated. Wistar rats subjected to critical sciatic nerve damage (12-mm gap) were divided into sham, autograft, PCL (empty NGC), and PCL + MSCs (NGC multi-functionalized with 106 canine AdMSCs embedded in heterologous fibrin biopolymer) groups. In vitro, the cells were characterized and directly stimulated with interferon-gamma to evaluate their neuroregeneration potential. In vivo, the sciatic and tibial functional indices were evaluated for 12 weeks. Gait analysis and nerve conduction velocity were analyzed after 8 and 12 weeks. Morphometric analysis was performed after 8 and 12 weeks following lesion development. Real-time PCR was performed to evaluate the neurotrophic factors BDNF, GDNF, and HGF, and the cytokine and IL-10. Immunohistochemical analysis for the p75NTR neurotrophic receptor, S100, and neurofilament was performed with the sciatic nerve. The inflammatory environment in vitro have increased the expression of neurotrophins BDNF, GDNF, HGF, and IL-10 in canine AdMSCs. Nerve guidance conduits multi-functionalized with canine AdMSCs embedded in HFB improved functional motor and electrophysiological recovery compared with PCL group after 12 weeks. However, the results were not significantly different than those obtained using autografts. These findings were associated with a shift in the regeneration process towards the formation of myelinated fibers. Increased immunostaining of BDNF, GDNF, and growth factor receptor p75NTR was associated with the upregulation of BDNF, GDNF, and HGF in the spinal cord of the PCL + MSCs group. A trend demonstrating higher reactivity of Schwann cells and axonal branching in the sciatic nerve was observed, and canine AdMSCs were engrafted at 30 days following repair. 3D-printed NGCs multi-functionalized with canine AdMSCs embedded in heterologous fibrin biopolymer as cell scaffold exerted neuroregenerative effects. Our multimodal approach supports the trophic microenvironment, resulting in a pro-regenerative state after critical sciatic nerve injury in rats.

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3D-printed nerve guidance conduits multi-functionalized with canine multipotent mesenchymal stromal cells promote neuroregeneration after sciatic nerve injury in rats.

Mesenchymal stem cell (MSC) is an absorbing candidate for cell therapy in treating spinal cord injury (SCI) due to its great potential for multiple cell differentiation, mighty paracrine secretion as well as vigorous immunomodulatory effect, of which are beneficial to the improvement of functional recovery post SCI. However, the therapeutic effects of MSC on SCI have been limited because of the gradual loss of MSC stemness in the process of expanding culture. Therefore, in this study, we aimed to maintain those beneficial properties of MSC via three-dimensional spheroid cell culture and then compared them with conventionally-cultured MSCs in the treatment of SCI both in vitro and in vivo with the aid of two-photon microscope. We found that 3D human placenta-derived MSCs (3D-HPMSCs) demonstrated a significant increase in secretion of anti-inflammatory factors and trophic factors like VEGF, PDGF, FGF via QPCR and Bio-Plex assays, and showed great potentials on angiogenesis and neurite morphogenesis when co-cultured with HUVECs or DRGs in vitro. After transplantation into the injured spinal cord, 3D-HPMSCs managed to survive for the entire experiment and retained their advantageous properties in secretion, and exhibited remarkable effects on neuroprotection by minimizing the lesion cavity, inhibiting the inflammation and astrogliosis, and promoting angiogenesis. Further investigation of axonal dieback via two-photon microscope indicated that 3D-HPMSCs could effectively alleviate axonal dieback post injury. Further, mice only treated with 3D-HPMSCs obtained substantial improvement of functional recovery on electrophysiology, BMS score, and Catwalk analysis. RNA sequencing suggested that the 3D-HPMSCs structure organization-related gene was significantly changed, which was likely to potentiate the angiogenesis and inflammation regulation after SCI. These results suggest that 3D-HPMSCs may hold great potential for the treatment of SCI.

3D spheroids of human placenta-derived mesenchymal stem cells attenuate spinal cord injury in mice.

Diabetic nephropathy (DN) is one of the most common diabetes mellitus (DM) microvascular complications, which always ends with end-stage renal disease (ESRD). Up to now, as the treatment of DN in clinic is still complicated, ESRD has become the main cause of death in diabetic patients. Mesenchymal stem cells (MSCs), with multi-differentiation potential and paracrine function, have attracted considerable attention in cell therapy recently. Increasing studies concerning the mechanisms and therapeutic effect of MSCs in DN emerged. This review summarizes several mechanisms of MSCs, especially MSCs derived exosomes in DN therapy, including hyperglycemia regulation, anti-inflammatory, anti-fibrosis, pro-angiogenesis, and renal function protection. We also emphasize the limitation of MSCs application in the clinic and the enhanced therapeutic role of pre-treated MSCs in the DN therapy. This review provides balanced and impartial views for MSC therapy as a promising strategy in diabetic kidney disease amelioration.

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The Multi-Therapeutic Role of MSCs in Diabetic Nephropathy.

Spinal cord injury (SCI) is a devasting condition with no reliable treatment. Spina bifida is the most common cause of congenital SCI. Cell-based therapies using mesenchymal stem/stromal cells (MSCS) have been largely utilized in SCI. Several clinical trials for acquired SCI use adult tissue-derived MSC sources, including bone-marrow, adipose, and umbilical cord tissues. The first stem/stromal cell clinical trial for spina bifida is currently underway (NCT04652908). The trial uses early gestational placental-derived mesenchymal stem/stromal cells (PMSCs) during the fetal repair of myelomeningocele. PMSCs have been shown to exhibit unique neuroprotective, angiogenic, and antioxidant properties, all which are promising applications for SCI. This review will summarize the unique properties and current applications of PMSCs and discuss their therapeutic role for acquired SCI.

The Unique Properties of Placental Mesenchymal Stromal Cells: A Novel Source of Therapy for Congenital and Acquired Spinal Cord Injury.

Abstract Background Inflammatory bowel diseases (IBD) are chronic relapsing–remitting inflammatory diseases of the gastrointestinal tract that are typically categorized into two subtypes: Crohn's disease (CD) and ulcerative colitis (UC). Although MSCs therapy has achieved encouraging outcomes in IBD therapy, objective responses are limited in colon fibrosis stenosis owing to the complicated microenvironment of CD and MSCs heterogeneity of quality. Here, we chose IFN-γ and kynurenic acid (KYNA) to overcome the low response and heterogeneity of human adipose-derived MSCs (hADSCs) to treat IBD and expand the therapeutic effects based on the excellent ability of IFN-γ and KYNA to promote indoleamine 2,3-dioxygenase-1 (IDO-1) signaling, providing a potential protocol to treat IBD and fibrosis disease. Methods hADSCs were isolated, cultured, and identified from human abdominal adipose tissue. The CD pathology-like acute colitis and chronic colon fibrosis rat model was induced by 2,4,6-trinitrobenzen sulfonic acid (TNBS). hADSCs were pretreated in vitro with IFN-γ and KYNA and then were transplanted intravenously at day 1 and 3 of TNBS administration in colitis along with at day 1, 15, and 29 of TNBS administration in chronic colonic fibrosis. Therapeutic efficacy was evaluated by body weights, disease activity index, pathological staining, real-time PCR, Western blot, and flow cytometry. For knockout of IDO-1, hADSCs were transfected with IDO-1-targeting small gRNA carried on a CRISPR-Cas9-lentivirus vector. Results hADSCs treated with IFN-γ and KYNA significantly upregulated the expression and secretion of IDO-1, which has effectively ameliorated CD pathology-like colitis injury and fibrosis. Notably, the ability of hADSCs with IDO-1 knockout to treat colitis was significantly impaired and diminished the protective effects of the primed hADSCs with IFN-γ and KYNA. Conclusion Inflammatory cytokines IFN-γ- and KYNA-treated hADSCs more effectively alleviate TNBS-induced colitis and colonic fibrosis through an IDO-1-dependent manner. Primed hADSCs are a promising new strategy to improve the therapeutic efficacy of MSCs and worth further research. 

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Therapeutic efficacy of human adipose mesenchymal stem cells in Crohn’s colon fibrosis is improved by IFN-γ and kynurenic acid priming through indoleamine 2,3-dioxygenase-1 signaling

Canine inflammatory brain disease (IBD) is a severe inflammatory disorder characterized by infiltration of activated immune cell subsets into the brain and spinal cord. Multipotent mesenchymal stromal cells (MSCs) are a promising therapy for IBD, based on their potent pro-angiogenic, neuroprotective, and immunomodulatory properties. The aims of this study were to compare the immunomodulatory attributes of canine adipose-derived MSCs (ASCs) and placenta-derived MSCs (PMSCs) in vitro. These data will serve as potency information to help inform the optimal MSC cell source to treat naturally occurring canine IBD. Indoleamine 2,3 dioxygenase (IDO) activity and prostaglandin E2 (PGE2) concentration at baseline and after stimulation with interferon gamma (IFNγ) and/or tumor necrosis factor alpha (TNFα) were measured from canine ASC and PMSC cultures. Leukocyte suppression assays (LSAs) were performed to compare the ability of ASCs and PMSCs to inhibit activated peripheral blood mononuclear cell (PBMC) proliferation. IDO activity and PGE2; interleukin (IL)-2, IL-6, and IL-8; TNFα; and vascular endothelial growth factor (VEGF) concentrations were also measured from co-culture supernatants. Cell cycle analysis was performed to determine how ASCs and PMSCs altered lymphocyte proliferation. Activated canine MSCs from both tissue sources secreted high concentrations of IDO and PGE2, after direct stimulation with IFNγ and TNFα, or indirect stimulation by activated PBMCs. Both ASCs and PMSCs inhibited activated PBMC proliferation in LSA assays; however, PMSCs inhibited PBMC proliferation significantly more than ASCs. Blocking PGE2 and IDO in LSA assays determined that PGE2 is important only for ASC inhibition of PBMC proliferation. Activated ASCs increased IL-6 and VEGF secretion and decreased TNFα secretion, while activated PMSCs increased IL-6, IL-8, and VEGF secretion. ASCs inhibited lymphocyte proliferation via cell cycle arrest in the G0/G1 and PMSCs inhibited lymphocyte proliferation via induction of lymphocyte apoptosis. Our results demonstrate that ASCs and PMSCs have substantial in vitro potential as a cell-based therapy for IBD; however, PMSCs more potently inhibited lymphocyte proliferation by inducing apoptosis of activated lymphocytes. These data suggest that the mechanism by which ASCs and PMSCs downregulate PBMC proliferation differs. Additional studies may elucidate additional mechanisms by which canine MSCs modulate neuroinflammatory responses.

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Rogério Martins Amorim

Papers

Placenta-derived multipotent mesenchymal stromal cells: a promising potential cell-based therapy for canine inflammatory brain disease