FGF-17 from Hypoxic Human Wharton's Jelly-Derived Mesenchymal Stem Cells Is Responsible for Maintenance of Cell Proliferation at Late Passages

BACKGROUND AND OBJECTIVES: Although it is well known that hypoxic culture conditions enhance proliferation of human mesenchymal stem cells, the exact mechanism is not fully understood. In this study, we investigated the effect of fibroblast growth factor (FGF)-17 from hypoxic human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs) on cell proliferation at late passages. METHODS AND RESULTS: hWJ-MSCs were cultured in α-MEM medium supplemented with 10% fetal bovine serum (FBS) in normoxic (21% O₂) and hypoxic (1% O₂) conditions. Protein antibody array was performed to analyze secretory proteins in conditioned medium from normoxic and hypoxic hWJ-MSCs at passage 10. Cell proliferation of hypoxic hWJ-MSCs was increased compared with normoxic hWJ-MSCs from passage 7 to 10, and expression of secretory FGF-17 was highly increased in conditioned medium from hypoxic hWJ-MSCs at passage 10. Knockdown of FGF-17 in hypoxic and normoxic hWJ-MSCs decreased cell proliferation, whereas treatment of hypoxic and normoxic hWJ-MSCs with recombinant protein FGF-17 increased their proliferation. Signal transduction of FGF-17 in hypoxic and normoxic hWJ-MSCs involved the ERK1/2 pathway. Cell phenotypes were not changed under either condition. Differentiation-related genes adiponectin, Runx2, and chondroadherin were downregulated in normoxic hWJ-MSCs treated with rFGF-17, and upregulated by siFGF-17. Expression of alkaline phosphatase (ALP), Runx2, and chondroadherin was upregulated in hypoxic hWJ-MSCs, and this effect was rescued by transfection with siFGF-17. Only chondroadherin was upregulated in hypoxic hWJ-MSCs with rFGF-17. CONCLUSIONS: In hypoxic culture conditions, FGF-17 from hypoxic hWJ-MSCs contributes to the maintenance of high proliferation at late passages through the ERK1/2 pathway.

The First Generation of iPSC Line from a Korean Alzheimer's Disease Patient Carrying APP-V715M Mutation Exhibits a Distinct Mitochondrial Dysfunction

Alzheimer's Disease (AD) is a progressive neurodegenerative disease, which is pathologically defined by the accumulation of amyloid plaques and hyper-phosphorylated tau aggregates in the brain. Mitochondrial dysfunction is also a prominent feature in AD, and the extracellular Aβ and phosphorylated tau result in the impaired mitochondrial dynamics. In this study, we generated an induced pluripotent stem cell (iPSC) line from an AD patient with amyloid precursor protein (APP) mutation (Val715Met; APP-V715M) for the first time. We demonstrated that both extracellular and intracellular levels of Aβ were dramatically increased in the APP-V715M iPSC-derived neurons. Furthermore, the APP-V715M iPSC-derived neurons exhibited high expression levels of phosphorylated tau (AT8), which was also detected in the soma and neurites by immunocytochemistry. We next investigated mitochondrial dynamics in the iPSC-derived neurons using Mito-tracker, which showed a significant decrease of anterograde and retrograde velocity in the APP-V715M iPSC-derived neurons. We also found that as the Aβ and tau pathology accumulates, fusion-related protein Mfn1 was decreased, whereas fission-related protein DRP1 was increased in the APP-V715M iPSC-derived neurons, compared with the control group. Taken together, we established the first iPSC line derived from an AD patient carrying APP-V715M mutation and showed that this iPSC-derived neurons exhibited typical AD pathological features, including a distinct mitochondrial dysfunction.

iPSC Modeling of Presenilin1 Mutation in Alzheimer's Disease with Cerebellar Ataxia

Disease modeling of Alzheimer's disease (AD) has been hampered by the lack of suitable cellular models while animal models are mainly based on the overexpression of AD-related genes which often results in an overemphasis of certain pathways and is also confounded by aging. In this study, we therefore developed and used induced pluripotent stem cell (iPSC) lines from a middle-aged AD patient with a known presenilin 1 (PSEN1) mutation (Glu120Lys; PS1-E120K) and as a control, an elderly normal subject. Using this approach, we demonstrated that the extracellular accumulation of Aβ was dramatically increased in PS1-E120K iPSC-derived neurons compared with the control iPSC line. PS1-E120K iPSC-derived neurons also exhibited high levels of phosphorylated tau, as well as mitochondrial abnormalities and defective autophagy. Given that the effect of aging is lost with iPSC generation, these abnormal cellular features are therefore indicative of PSEN1-associated AD pathogenesis rather than primary changes associated with aging. Taken together, this iPSC-based approach of AD modeling can now be used to better understand AD pathogenesis as well as a tool for drug discovery.

Effect of Single and Double Administration of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Following Focal Cerebral Ischemia in Rats

Stem cell therapies are administered during the acute phase of stroke to preserve the penumbral tissues from ischemic injury. However, the effect of repeated cell therapy during the acute phase remains unclear. In this study, we investigated and compared the functional outcome of single (two days post-injury) and repeated (two and nine days post-injury) treatment with human umbilical cord derived mesenchymal stem cells (hUCB-MSCs) after middle cerebral artery occlusion (MCAO). The rotarod and limb placement tests were utilized to investigate functional outcomes, while infarct volume and tissue damage were measured by immunofluorescent staining for neovascularization, neurogenesis, apoptosis, and inflammation in the penumbral zones. We observed notable motor dysfunction and a significant decrease in infarcted brain volume, as well as increases in neurons and vessels in both single and repeated hUCB-MSC treatments compared to the control group. Interestingly, repeated administration of hUCB-MSCs was not found to elicit additional or synergistic improvements over monotherapy. This study suggests that a clearer understanding of the therapeutic window after stroke will facilitate the development of more efficient treatment protocols in the clinical application of stem cell therapy.

The Effect of Donor-Dependent Administration of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells following Focal Cerebral Ischemia in Rats

Stroke is an ischemic disease caused by clotted vessel-induced cell damage. It is characterized by high morbidity and mortality and is typically treated with a tissue plasminogen activator (tPA). However, this therapy is limited by temporal constraints. Recently, several studies have focused on cell therapy as an alternative treatment. Most researches have used fixed donor cell administration, and hence, the effect of donor-dependent cell administration is unknown. In this study, we administered 3 types of donor-derived human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) in the ischemic boundary zone of the ischemic stroke rat model. We then performed functional and pathological characterization using rotarod, the limb placement test, and immunofluorescent staining. We observed a significant decrease in neuron number, and notable stroke-like motor dysfunction, as assessed by the rotarod test (~40% decrease in time) and the limb placement test (4.5 point increase) in control rats with ischemic stroke. The neurobehavioral performance of the rats with ischemic stroke that were treated with hUCB-MSCs was significantly better than that of rats in the vehicle-injected control group. Regardless of which donor cells were used, hUCB-MSC transplantation resulted in an accumulation of neuronal progenitor cells, and angiogenic and tissue repair factors in the ischemic boundary zone. The neurogenic and angiogenic profiles of the 3 types of hUCB-MSCs were very similar. Our results suggest that intraparenchymal administration of hUCB-MSCs results in significant therapeutic effects in the ischemic brain regardless of the type of donor.

The Effect of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells in a Collagenase-Induced Intracerebral Hemorrhage Rat Model

Intracerebral hemorrhage (ICH) is one of the devastating types of stroke. Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have potential benefits in recovery from brain damage following ICH. This study aimed to identify the beneficial effects of hUCB-MSCs and investigate whether they have anti-inflammatory effects on the ICH brain via neurotrophic factors or cytokines. hUCB-MSCs were transplanted into a collagenase-induced ICH rat model. At 2, 9, 16, and 30 days after ICH, rotarod and limb placement tests were performed to measure behavioral outcomes. ICH rats were sacrificed to evaluate the volume of lesion using H&E staining. Immunostaining was performed to investigate neurogenesis, angiogenesis, and anti-apoptosis at 4 weeks after transplantation. Inflammatory factors (TNF-alpha, COX-2, microglia, and neutrophils) were analyzed by immunofluorescence staining, RT-PCR, and Western blot at 3 days after transplantation. hUCB-MSCs were associated with neurological benefits and reduction in lesion volume. The hUCB-MSCs-treated group tended to reveal high levels of neurogenesis, angiogenesis, and anti-apoptosis (significant for angiogenesis). The expression levels of inflammatory factors tended to be reduced in the hUCB-MSCs-treated group compared with the controls. Our study suggests that hUCB-MSCs may improve neurological outcomes and modulate inflammation-associated immune cells and cytokines in ICH-induced inflammatory responses.

Recent Trends and Strategies in Stem Cell Therapy for Alzheimer's Disease / 한양의대학술지

The human mesenchymal stem cell (MSC) has been regarded as a fascinating candidate of stem cell therapy in neurodegenerative disorders such as Alzheimer disease (AD). Recently, many groups reported that mesenchymal stem cell is a robust source, not only of regeneration, but also of secretion of various soluble factors for damaged or lost host cells. Several groups have observed paracrine action of mesenchymal stem cells in transgenic mice models of AD. From non-clinical studies we can conclude that human mesenchymal stem cells could participate in anti-apoptosis, beta-amyloid removal, anti-inflammation and anti-tau aggregation via paracrine action. Based on these findings, several clinical trials have been performed or completed worldwide. Since safety and efficacy have been confirmed from various non-clinical and clinical trials, we can expect emerging use of stem cells for AD in the near future.