Repeat intramuscular transplantation of human dental pulp stromal cells is more effective in sustaining Schwann cell survival and myelination for functional recovery after onset of diabetic neuropathy.

BACKGROUND AIMS: Mesenchymal stromal cell (MSC) therapy for diabetic neuropathy (DN) has been extensively researched in vitro and in pre-clinical studies; however, the clinical scenario thus far has been disappointing. Temporary recovery, a common feature of these studies, indicates that either the retention of transplanted cells deteriorates with time or recovery of supportive endogenous cells, such as bone marrow-derived MSCs (BM-MSCs), does not occur, requiring further replenishment. In DN, BM-MSCs are recognized mediators of Schwann cell regeneration, and we have earlier shown that they suffer impairment in the pre-neuropathy stage. In this study, we attempted to further elucidate the mechanisms of functional recovery by focusing on changes occurring at the cellular level in the sciatic nerve, in conjunction with the biodistribution and movement patterns of the transplanted cells, to define the interval between doses. METHOD & RESULTS: We found that two doses of 1 × 106 dental pulp stromal cells (DPSCs) transplanted intramuscularly at an interval of 4 weeks effectively improved nerve conduction velocity (NCV) and restored motor coordination through improving sciatic nerve architecture, Schwann cell survival and myelination. Despite very minimal recovery of endogenous BM-MSCs, a temporary restoration of NCV and motor function was achieved with the first dose of DPSC transplantation. However, this did not persist, and a repeat dose was needed to consolidate functional improvement and rehabilitate the sciatic nerve architecture. CONCLUSION: Thus, repeat intramuscular transplantation of DPSCs is more effective for maintenance of Schwann cell survival and myelination for functional recovery after onset of DN.

Interventional Strategies for Parkinson Disease: Can Neural Precursor Cells Forge a Path Ahead?

Neural precursor cells (NPCs), derived from pluripotent stem cells (PSCs), with their unique ability to generate multiple neuronal and glial cell types are extremely useful for understanding biological mechanisms in normal and diseased states. However, generation of specific neuronal subtypes (mature) from NPCs in large numbers adequate for cell therapy is challenging due to lack of a thorough understanding of the cues that govern their differentiation. Interestingly, neural stem cells (NSCs) themselves are in consideration for therapy given their potency to form different neural cell types, release of trophic factors, and immunomodulatory effects that confer neuroprotection. With the recent COVID-19 outbreak and its accompanying neurological indications, the immunomodulatory role of NSCs may gain additional significance in the prevention of disease progression in vulnerable populations. In this regard, small-molecule mediated NPC generation from PSCs via NSC formation has become an important strategy that ensures consistency and robustness of the process. The development of the mammalian brain occurs along the rostro-caudal axis, and the establishment of anterior identity is an early event. Wnt signaling, along with fibroblast growth factor and retinoic acid, acts as a caudalization signal. Further, the increasing amount of epigenetic data available from human fetal brain development has enhanced both our understanding of and ability to experimentally manipulate these developmental regulatory programs in vitro. However, the impact on homing and engraftment after transplantation and subsequently on therapeutic efficacy of NPCs based on their derivation strategy is not yet clear. Another formidable challenge in cell replacement therapy for neurodegenerative disorders is the mode of delivery. In this Perspective, we discuss these core ideas with insights from our preliminary studies exploring the role of PSC-derived NPCs in rat models of MPTP-induced Parkinson's disease following intranasal injections.

Fundamental changes in endogenous bone marrow mesenchymal stromal cells during Type I Diabetes is a pre-neuropathy event.

Deficiency of angiogenic and neurotrophic factors under long term diabetes is known to lead to Schwann cell degeneration, clinically manifested as Diabetic Neuropathy (DN). While the transplantation of exogenous allogenic Mesenchymal Stromal Cells (MSCs) has shown amelioration of DN through paracrine action, it is not known what functional changes occur in endogenous bone-marrow MSCs under chronic diabetes in terms of homing, migration and/or paracrine signalling with reference to the end-point clinical manifestation of Diabetic Neuropathy. We thus aimed at determining the changes in BM-MSCs under Type 1 Diabetes with respect to survival, self-renewal, oxidative status, paracrine activity, intracellular Ca2+ response and migration in response to pathological cytokine/chemokine, in reference to the time-point of decline in Nerve Conduction Velocity (NCV) in a rat model. Within one week of diabetes induction, BM-MSCs underwent apoptosis, and compromised their self-renewal capacity, antioxidant defence mechanism and migration toward cytokine/chemokine; whereas epineurial blood vessel thickening and demyelination resulting in NCV decline were observed only after three weeks. By two- and three-weeks post diabetes induction, BM-MSC apoptosis reduced and proliferative ability was restored; however, their self-renewal, migration and intracellular Ca2+ response toward pathological cytokine/chemokine remained impaired. These results indicate that T1D induced intrinsic functional impairments in endogenous BM-MSCs occur before neuropathy onset. This timeline of functional alterations in BM-MSCs also suggest that treatment strategies that target the bone marrow niche early on may help to modulate BM-MSC functional impairments and thus slow down the progression of neuropathy.

Mesenchymal stromal cell therapy for coronavirus disease 2019: which? when? and how much?

Mesenchymal stromal cells (MSCs) are under active consideration as a treatment strategy for controlling the hyper-inflammation and slow disease progression associated with coronavirus disease 2019 (COVID-19). The possible mechanism of protection through their immunoregulatory and paracrine action has been reviewed extensively. However, the importance of process control in achieving consistent cell quality, maximum safety and efficacy-for which the three key questions are which, when and how much-remains unaddressed. Any commonality, if it exists, in ongoing clinical trials has yet to be analyzed and reviewed. In this review, the authors have therefore compiled study design data from ongoing clinical trials to address the key questions of "which" with regard to tissue source, donor profile, isolation technique, culture conditions, long-term culture and cryopreservation of MSCs; "when" with regard to defining the transplantation window by identifying and staging patients based on their pro-inflammatory profile; and "how much" with regard to the number of cells in a single administration, number of doses and route of transplantation. To homogenize MSC therapy for COVID-19 on a global scale and to make it readily available in large numbers, a shared understanding and uniform agreement with respect to these fundamental issues are essential.

Human bone marrow-derived, pooled, allogeneic mesenchymal stromal cells manufactured from multiple donors at different times show comparable biological functions in vitro, and in vivo to repair limb ischemia.

BACKGROUND: We have previously demonstrated that a pooled population of bone marrow-derived, allogeneic mesenchymal stromal cells (BMMSC), Stempeucel®-1, produced under good manufacturing practices (GMP) conditions, showed clinical efficacy and safety in patients suffering from critical limb ischemia (CLI) due to Buerger's disease. While Stempeucel®-1 is currently used for CLI and other clinical indications, we wanted to ensure that the product's continuity is addressed by developing and characterizing a second generation of pooled product (Stempeucel®-1A), manufactured identically from second BM aspirates of the same three donors after a 2-year interval. METHODS: The two versions of Stempeucel® were manufactured and subjected to gene and protein expression analysis. The nature of various growth factors/cytokines secreted and immunomodulatory activity of these two cell populations were compared directly by various in vitro assays. The preclinical efficacy of these two cell types was compared in an experimental model of hind limb ischemia (HLI) in BALB/c nude mice. The reversal of ischemia, blood flow, and muscle regeneration were determined by functional scoring, laser Doppler imaging, and immunohistochemical analyses. RESULTS: Qualitative and quantitative analyses of genes and proteins involved in promoting angiogenic activity and immune regulatory functions revealed high levels of correlation between Stempeucel®-1 and Stempeucel®-1A cell populations. Moreover, intramuscular (i.m) administration of these two cell products in the ischemic limbs of BALB/c nude mice showed significant repair (≥ 70%) of toe and foot necrosis, leading to improved ambulatory function and limb salvage. Furthermore, a biodistribution kinetics study showed that Stempeucel®-1 was mostly localized in the ischemic muscles of mice for a significantly longer time compared to normal muscles, thus playing an essential role in modulating and reversing HLI damage. CONCLUSIONS: This study shows that with a reproducible manufacturing procedure, it is possible to generate large numbers of pooled mesenchymal stromal cells from human bone marrow samples to establish product equivalence. We conclude from these results that, for the first time, two pooled, allogeneic BMMSC products can be repeatedly manufactured at different time intervals using a two-tier cell banking process with robust and comparable angiogenic properties to treat ischemic diseases.

Biodistribution of Intramuscularly-Transplanted Human Dental Pulp Stem Cells in Immunocompetent Healthy Rats through NIR Imaging.

Owing to their neural crest origin, dental pulp stem cells (DPSCs) are increasingly gaining prominence in treating nervous system disease conditions. However, as per the regulatory bodies [European-Medicines Agency (EMA), Indian-Council of Medical-Research (ICMR)], their biodistribution after transplantation needs to be evaluated for them to be considered for cell-based therapy for clinical trials. There are yet no studies describing the dynamic distribution of human origin DPSCs (hDPSCs) after transplantation in an immunocompetent, physiologically healthy animal model. Here, using near-infrared (NIR)-based whole animal and ex vivo tissue imaging, we assessed the biodistribution of intramuscularly transplanted hDPSCs in immunocompetent healthy Wistar rats. Further validation was done by quantifying gene expression of the human Alu gene in rat tissues. After 24 h of transplantation, an increase in signal intensity and area of signal was observed in the muscle of administration compared to 30 min and 6 h. At hour 24, neither increase in human Alu nor human Ki67 gene expression was seen in the rat muscle, thus confirming that the increase in signal area and intensity at hour 24 was not due to proliferation of the transplanted cells. Rather at hour 24, the NIR-signal intensity in bone marrow increased, suggesting that the NIR-tagged DPSCs have started entering into the blood vessels adjacent to the muscle, and the blood vessels being placed just beneath the subcutaneous layer might be responsible for an increase in signal intensity. Signal intensity increased distinctly in all organs at this timepoint, confirming that the cells entered the bloodstream by hour 24. Lung entrapment of DPSCs was not observed, since signal intensity was least in lungs as compared to the site of injection. Cells were retained for up to 28 days at the site of injection. These findings lay the basis to design the dosage for intramuscular delivery of hDPSCs for degenerative disease models and for future clinical trials.

Functional recovery upon human dental pulp stem cell transplantation in a diabetic neuropathy rat model.

Diabetic neuropathy (DN) is among the most debilitating complications of diabetes. Here, we investigated the effects of human dental pulp stem cell (DPSC) transplantation in Streptozotocin (STZ)-induced neuropathic rats. Six weeks after STZ injection, DPSCs were transplanted through two routes, intravenous (IV) or intramuscular (IM), in single or two repeat doses. Two weeks after transplantation, a significant improvement in hyperalgesia, grip-strength, motor coordination and nerve conduction velocity was observed in comparison with controls. A rapid improvement in neuropathic symptoms was observed for a single dose of DPSC IV; however, repeat dose of DPSC IV did not bring about added improvement. A single dose of DPSC IM showed steady improvement, and further recovery continued upon repeat IM administration. DPSC single dose IV showed greater improvement than DPSC single dose IM, but IM transplantation brought about better improvement in body weight. A marked reduction in tumor necrosis factor (TNF) α and C-reactive protein (CRP) levels was observed in the blood plasma for all treated groups, as compared with controls. With respect to inflammatory cytokines, repeat dose of DPSC IM showed further improvement, suggesting that a repeat dose is required to maintain the improved inflammatory state. Gene expression of inflammatory markers in liver confirmed amelioration in inflammation. Arachidonic acid level was unaffected by IV DPSC transplantation but showed noticeable increase through IM administration of a repeat dose. These results suggest that DPSC transplantation through both routes and dosage was beneficial for the retrieval of neuropathic parameters of DN; transplantation via the IM route with repeat dose was the most effective.

Generation of islet-like cell aggregates from human non-pancreatic cancer cell lines.

To explore a novel source for the derivation of islets, we examined the differentiation potential of human non-pancreatic cancer cell lines, HeLa (cervical carcinoma cell line) and MCF-7 (breast cancer cell line). These cells were subjected to a serum-free, three-step sequential differentiation protocol which gave two distinct cell populations: single cells and cellular aggregates. Subsequent analysis confirmed their identity as pancreatic acinar cells and islet-like cell aggregates (ICAs), as evidenced by amylase secretion and diphenylthiocarbazone staining respectively. Reverse transcriptase-PCR and immunocytochemistry assessment of the ICAs revealed the expression of pancreatic specific markers Ngn-3, Glut-2, Pax-6 and Isl-1. These ICAs secreted insulin in response to glucose challenge, confirming their functionality. We propose that ICAs generated from HeLa and MCF-7 cell lines could form a promising in vitro platform of human islet equivalents (hIEQs) for diabetes research.