Development of Adipose-Derived Stromal/stem Cell–Based Therapies: Effects of donor weight and culture conditions on cell characteristics and testing the efficacy in stroke models

Cell-based therapies are novel methods to treat medical conditions that have limited or no effective therapeutic options such as ischemic stroke. In particular, adipose-derived stromal/stem cells (ASCs) are promising cell types for various clinical applications because of their proliferation and multilineage differentiation capacity, their low immunogenicity and especially their immunomodulation ability. Via paracrine effects, ASCs facilitate tissue regeneration by reducing apoptosis and scarring and by increasing angiogenesis. Before ASCs can be used in clinical practice, the effects of donor characteristics need to be studied, ASC culture conditions need to be optimized, and the safety and efficacy of ASCs must be verified both in vitro and in vivo. In this thesis we first studied the effect of donor body mass index (BMI) and defined, xeno-free and serum-free (XF/SF) culture conditions on ASC characteristics. After that the efficacy of ASC therapy for ischemic stroke was evaluated in vitro and in vivo.

The success of future cell-based therapies is linked to the appropriate selection of cell donors. Several factors, such as donor age, sex and location of the harvested adipose tissue, affect ASC characteristics. ASC characteristics have been shown to change depending on BMI. Obesity and high BMI are associated with chronic low- grade inflammation and altered metabolic and endocrine functions in adipose tissue. In the first study, the effects of donor weight on ASC characteristics, including proliferation, immunophenotype, differentiation capacity, immunogenicity, immunosuppression capacity and angiogenic potential, were studied. ASCs were isolated from genetically matched monozygotic weight-discordant twin pairs (ΔBMI > 3 kg/m2). The results showed that an increased donor weight did not affect immunogenicity, proliferation, or osteogenic capacity. However, adipogenic differentiation capacity, the expression of proinflammatory markers such as TNF and immunosuppression capacity increased, while CD146 expression and angiogenic potential decreased, with increased donor weight.

Prior to clinical cell therapies, cells are isolated and expanded in vitro, for which animal-derived (xenogeneic) reagents, such as fetal bovine serum, have commonly been used. The use of xenogeneic reagents poses a risk of immunological reactions as well as zoonoses, which are potentially transferred into the patient upon cell transplantation and lot-to-lot variations in medium components, such as serum, may cause variations in cell behavior. In the second study, XF/SF isolation and culture protocol for ASCs in which ASCs maintained their differentiation capacity, ASC immunophenotype and proliferation capacity was developed.

Cell-based therapies are some of the most attractive strategies to enhance recovery after ischemic stroke. Human ASCs have been shown to secrete angiogenic and neurogenic factors. In the third study, neuronally differentiated neuroblastoma cells (SH-SY5Y cells) and induced pluripotent stem cells (hiPSCs) were used to model stroke in vitro with oxygen-glucose deprivation (OGD). With these developed models, the paracrine effects of ASCs on neuronal cell recovery were investigated. OGD induced cytotoxicity and apoptosis of SH-SY5Y and hiPSC neurons; however, the damage was more severe in SH-SY5Y neurons than in hiPSC neurons. ASC coculture decreased apoptosis and increased proliferation of neurons from both cell types.

In the fourth study, the effect of combined ASC therapy and rehabilitation in an enriched environment was studied after stroke injury in a rat model. In the study, a brain vein was closed, and local damage in the cerebral cortex developed. Human ASCs were injected into the rat tail vein two or seven days after damage. The results showed that ASCs alone and rehabilitation alone moderately enhanced stroke recovery according to sensorimotor tests. The combination therapy further improved sensorimotor functions. In this study, angiogenesis and gliosis were investigated as the mechanisms of action of combined therapy; however, the improved recovery was not related to the studied mechanisms.

In this research, we found that the weight of the donor influences ASC characteristics and therefore should be considered when donors are selected for ASC therapies. We found that ASCs can be cultured under novel XF/SF conditions and that they retain their typical characteristics under XF/SF conditions. ASC coculture showed neuroprotective effects in in vitro stroke models and the combination of ASC therapy and rehabilitation improved stroke recovery in vivo. More preclinical studies and clinical trials are needed to ensure the efficacy and clarify the mechanisms of action of ASC therapy for ischemic stroke.