Aging restricts the ability of mesenchymal stem cells to promote the generation of oligodendrocytes during remyelination.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) that leads to severe neurological deficits. Due to their immunomodulatory and neuroprotective activities and their ability to promote the generation of oligodendrocytes, mesenchymal stem cells (MSCs) are currently being developed for autologous cell therapy in MS. As aging reduces the regenerative capacity of all tissues, it is of relevance to investigate whether MSCs retain their pro-oligodendrogenic activity with increasing age. We demonstrate that MSCs derived from aged rats have a reduced capacity to induce oligodendrocyte differentiation of adult CNS stem/progenitor cells. Aging also abolished the ability of MSCs to enhance the generation of myelin-like sheaths in demyelinated cerebellar slice cultures. Finally, in a rat model for CNS demyelination, aging suppressed the capability of systemically transplanted MSCs to boost oligodendrocyte progenitor cell (OPC) differentiation during remyelination. Thus, aging restricts the ability of MSCs to support the generation of oligodendrocytes and consequently inhibits their capacity to enhance the generation of myelin-like sheaths. These findings may impact on the design of therapies using autologous MSCs in older MS patients.

Selection of Tissue Factor-Deficient Cell Transplants as a Novel Strategy for Improving Hemocompatibility of Human Bone Marrow Stromal Cells.

Intravascular transplantation of tissue factor (TF)-bearing cells elicits an instant blood-mediated inflammatory reaction (IBMIR) resulting in thrombotic complications and reduced engraftment. Here we studied the hemocompatibility of commonly used human white adipose tissue (WAT), umbilical cord (UC) and bone marrow stromal cells (BMSC) and devised a possible strategy for safe and efficient stromal cell transplantation. Methods: Stromal cell identity, purity, and TF expression was tested by RTQ-PCR, flow cytometry and immunohistochemistry. Pro-coagulant activity and fibrin clot formation/stabilization was measured In Vitro by viscoelastic rotational plasma-thromboelastometry and in vivo by injecting sorted human stromal cells intravenously into rats. The impact of TF was verified in factor VII-deficient plasma and by sort-depleting TF/CD142+ BMSC. Results: We found significantly less TF expression by a subpopulation of BMSC corresponding to reduced pro-coagulant activity. UC and WAT stroma showed broad TF expression and durable clotting. Higher cell numbers significantly increased clot formation partially dependent on coagulation factor VII. Depleting the TF/CD142+ subpopulation significantly ameliorated BMSC's hemocompatibility without affecting immunomodulation. TF-deficient BMSC did not produce thromboembolism in vivo, comparing favorably to massive intravascular thrombosis induction by TF-expressing stromal cells. Conclusion: We demonstrate that plasma-based thromboelastometry provides a reliable tool to detect pro-coagulant activity of therapeutic cells. Selecting TF-deficient BMSC is a novel strategy for improving cell therapy applicability by reducing cell dose-dependent IBMIR risk. The particularly strong pro-coagulant activity of UC and WAT preparations sounds an additional note of caution regarding uncritical systemic application of stromal cells, particularly from non-hematopoietic extravascular sources.

A Good Manufacturing Practice-grade standard protocol for exclusively human mesenchymal stromal cell-derived extracellular vesicles.

BACKGROUND AIMS: Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) may contribute to biological processes such as tissue regeneration, immunomodulation and neuroprotection. Evaluation of their therapeutic potential and application in future clinical trials demands thorough characterization of EV content and production under defined medium conditions, devoid of xenogenic substances and serum-derived vesicles. Addressing the apparent need for such a growth medium, we have developed a medium formulation based on pooled human platelet lysate (pHPL), free from animal-derived xenogenic additives and depleted of EVs. METHODS: Depletion of EVs from complete growth medium was achieved by centrifugation at 120 000 g for 3 h, which reduced RNA-containing pHPL EVs to below the detection limit. RESULTS: Bone marrow (BM)-derived MSCs propagated in this medium retained the characteristic surface marker expression, cell morphology, viability and in vitro osteogenic and adipogenic differentiation potential. The proliferation rate was not significantly affected after 48 h but was decreased by 13% after 96 h. EVs collected from BM-MSCs cultured in EV-depleted medium revealed a similar RNA pattern as EVs generated in standard pHPL EV-containing medium but displayed a more clearly defined pattern of proteins characteristic for EVs. Reduction of pHPL content from 10% to 2% or serum-/pHPL-free conditions strongly altered MSC characteristics and RNA content of released EV. CONCLUSIONS: The 10% pHPL-based EV-depleted medium is appropriate for purification of exclusively human MSC-derived EVs. With this Good Manufacturing Practice-grade protocol, characterization and establishment of protein and RNA profiles from MSC-derived EVs can now be achieved to identify active components in therapeutic EVs for future clinical application.

Lesion-induced accumulation of platelets promotes survival of adult neural stem / progenitor cells.

The presence of neural stem/progenitor cells (NSPCs) in specific areas of the central nervous system (CNS) supports tissue maintenance as well as regeneration. The subependymal zone (SEZ), located at the lateral ventricle's wall, represents a niche for NSPCs and in response to stroke or demyelination becomes activated with progenitors migrating towards the lesion and differentiating into neurons and glia. The mechanisms that underlie this phenomenon remain largely unknown. The vascular niche and in particular blood-derived elements such as platelets, has been shown to contribute to CNS regeneration in different pathological conditions. Indeed, intracerebroventricularly administrated platelet lysate (PL) stimulates angiogenesis, neurogenesis and neuroprotection in the damaged CNS. Here, we explored the presence of platelets in the activated SEZ after a focal demyelinating lesion in the corpus callosum of mice and we studied the effects of PL on proliferating SEZ-derived NSPCs in vitro. We showed that the lesion-induced increase in the size of the SEZ and in the number of proliferating SEZ-resident NSPCs correlates with the accumulation of platelets specifically along the activated SEZ vasculature. Expanding on this finding, we demonstrated that exposure of NSPCs to PL in vitro led to increased numbers of cells by enhanced cell survival and reduced apoptosis without differences in proliferation and in the differentiation potential of NSPCs. Finally, we demonstrate that the accumulation of platelets within the SEZ is spatially correlated with reduced numbers of apoptotic cells when compared to other periventricular areas. In conclusion, our results show that platelet-derived compounds specifically promote SEZ-derived NSPC survival and suggest that platelets might contribute to the enlargement of the pool of SEZ NSPCs that are available for CNS repair in response to injury.