Neuronal differentiation of adipose-derived stem cells and their transplantation for cerebral ischemia.

OBJECTIVE: To review published data on the biological characteristics, differentiation and applications of adipose-derived stem cells in ischemic diseases. DATA RETRIEVAL: A computer-based online search of reports published from January 2005 to June 2012 related to the development of adipose-derived stem cells and their transplantation for treatment of cerebral ischemia was performed in Web of Science using the key words "adipose-derived stem cells", "neural-like cells", "transplantation", "stroke", and "cerebral ischemia". SELECTION CRITERIA: The documents associated with the development of adipose-derived stem cells and their transplantation for treatment of cerebral ischemia were selected, and those published in the last 3-5 years or in authoritative journals were preferred in the same field. Totally 89 articles were obtained in the initial retrieval, of which 53 were chosen based on the inclusion criteria. MAIN OUTCOME MEASURES: Biological characteristics and induced differentiation of adipose-derived stem cells and cell transplantation for disease treatment as well as the underlying mechanism of clinical application.
RESULTS: The advantages of adipose-derived stem cells include their ease of procurement, wide availability, rapid expansion, low tumorigenesis, low immunogenicity, and absence of ethical constraints. Preclinical experiments have demonstrated that transplanted adipose-derived stem cells can improve neurological functions, reduce small regions of cerebral infarction, promote angiogenesis, and express neuron-specific markers. The improvement of neurological functions was demonstrated in experiments using different methods and time courses of adipose-derived stem cell transplantation, but the mechanisms remain unclear.
CONCLUSION: Further research into the treatment of ischemic disease by adipose-derived stem cell transplantation is needed to determine their mechanism of action.

Research Highlights

Adipose-derived stem cells (ADSCs) are ideal candidates for neural stem cell transplantation.

This study summarizes studies on ADSCs transplantation for cerebral ischemia with respect to the biological characteristics of ADSCs, the expression of surface antigens by ADSCs, the differentiation of ADSCs into nerve cells, stereotaxic heterogenic transplantation of ADSCs, heterogenic intravenous transplantation of ADSCs, and allogeneic intravenous transplantation of ADSCs. The transplantation of ADSC-conditioned medium for the treatment of cerebrovascular disease is also discussed.

Abbreviations

ADSCs, adipose-derived stem cells; MSCs, mesenchymal stem cells; EGF, epidermal growth factor; FGF, fibroblast growth factor; MCAO: middle cerebral artery occlusion

INTRODUCTION

Cerebral ischemia is a common acute cerebrovascular disease. The key to its treatment involves the rescue of dying neurons in the ischemic penumbra and the promotion of recovery from injury. The accepted mechanisms for cerebral ischemia include: Ca2+ overloading, free radical and lipid peroxidation, mitochondrial dysfunction, deregulated expression of nitrogen monoxide, cytokines, immediate early genes, heat shock protein, and cell apoptosis. Cerebral ischemia is associated with high levels of mortality and disability, and current medical therapy for cerebral ischemia patients is limited. Most patients primarily receive supportive treatment[1] and only selected patients are subjected to surgical treatment[2]. Neural cell replacement therapy is the only effective treatment. Neural cell transplantation can rebuild nerve conduction loops and restore some neurological function, possibly via the differentiation of transplanted stem cells into functional glial cells or neurons, which can thus substitute for some of the affected neurons. The transplanted stem cells secrete cytokines to improve the local microenvironment of ischemic necrosis in terms of inflammation, tissue necrosis and glial scarring[34]. However, cerebral ischemic damage can affect large areas and many kinds of nerve cells. Ischemic lesions may involve a number of sites in the hypothalamus, striatum, hippocampus and cortex, and reconstruction of this complex system presents a challenge for cell transplantation. Mesenchymal stem cells (MSCs) differ from hematopoietic stromal cells in that they are pluripotent precursor cells. Adipose-derived MSCs (ADSCs) are similar to marrow stromal cells, and offer advantages such as strong amplification, convenience and availability, and lack of immune rejection. As such, ADSCS have proven to be a feasible and effective cell source for neural cell replacement therapy in cerebral ischemia[56].

DATA SOURCES AND METHODOLOGY

Data retrieval

A computer-based online search of original reports published from January 2005 to June 2012 related to the development of ADSCs and their transplantation for treatment of cerebral ischemia was performed in Web of Science using the key words “adipose-derived stem cells”, “adipose stem cells”, “differentiation”, “neural-like cells”, “transplantation”, “brain stroke”, and “cerebral ischemia”.

Selection criteria

The documents associated with the development of ADSCs and their transplantation for treatment of cerebral ischemia were selected, and those published in the last five years or in authoritative journals were preferred in the same field. Repetitive studies were excluded from this review. 89 articles in English and Chinese were obtained in the initial retrieval, of which 53 were chosen for review based on the inclusion criteria. A flow chart summarizing the literature screening process is shown below:

RESULTS

Biological characteristics of ADSCs

In 2001, researchers from the University of California Los Angeles and the University of Pittsburgh first reported that stem cells could be isolated from adipose tissue removed during liposuction[7]. Since then, several groups have reported the isolation of stem cells from adipose tissues by similar isolation methods[89]. Recently, researchers have suggested that ADSCs can be differentiated into new fat tissue, bone, cartilage, nerve, muscle and endothelial cells[1011]. In animal studies, these cells show potential for the treatment of heart attack, stroke, bone injury, and even neurological diseases[1213]. ADSCs have been referred to as preadipocytes, mesenchymal cells, treated liposuction cells, multi-potential adipose-derived stem cells and adipose-derived adult stromal cells, and were named adipose-derived stem cells at the 2nd annual meeting of the International Fat Applied Technology Society in 2004[1415].

Expression of surface antigens by ADSCs

ADSCs possess the same characteristics as MSCs[16171819], including (1) adhesion in routine culture conditions; (2) the potential to differentiate into bone, fat and cartilage; (3): a similar morphology to fibroblasts, with adhesive polymeric colony-forming units; (4) positive expression of Stro-1, CD13, CD29, CD44, CD63, CD73, CD90 and CD166. The expression of these markers is low during the early stages of culture and increases significantly with increasing passage number[2021]; (5) negative expression of hemopoietic stem cell markers such as c-kit, CD11b, CD14, CD19, CD34, CD45, CD79a and HLA-DR. In general, the markers for ADSCs are different in each experiment, possibly because of different culture methods, different cell passage numbers, distinct immunohistochemical susceptibility or diverse monoclonal antibodies. Currently, there are no specific surface markers for adipose-derived stem cells. In experiments to characterize ADSCs, 3–5 representative positive or negative markers are selected to identify the ADSCs. Flow cytometry in particular, and also immunohistochemistry, are commonly used to identify ADSCs.

Induced differentiation of ADSCs into nerve cells

With the maturation of techniques for the in vitro differentiation of ADSCs, researchers have investigated whether ADSCs can survive and differentiate in vivo. Kopen et al[22] injected bone marrow stromal stem cells into the lateral cerebral ventricles of mice, and 12 days later the cells had migrated to the olfactory bulb, procerebrum and cerebellum, and some cells had migrated into the corpora striata and stratum moleculare hippocampi where they expressed glial fibrillary acidic protein. Other cells migrated to the reticular formation of the brain stem and expressed NF-70. This migration is similar to the process of postnatal neural development, which indicates that as neural precursor cells, bone marrow stromal stem cells can migrate in the brain following transplantation into the lateral cerebral ventricle and differentiate into glioblasts and neurons.

Safford et al[23] injected induced ADSCs into the hippocampal CA1 region and found that the transplanted ADSCs migrated along the corpus callosum and corpus striatum from the mouth and tail shaft for up to 2 mm, and survived for 12 weeks. The induced ADSCs resembled nerve cells due to the expression of neurone specific enolase and NeuN. However, ADSCs that were not induced in vitro did not exhibit these behaviors, indicating that in vitro induction of ADSCs is essential for their survival in the microenvironment after transplantation.

Many factors influence the neural differentiation and migration of ADSCs, such as the chemokines epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), insulin-like growth factor, platelet-derived grow factor, brain-derived neurotrophic factor and nerve growth factor, which have been demonstrated to promote proliferation and neurogenesis in stem cells in the mammalian brain. EGF is the most potent stimulator of neurogenesis in neural stem cells in the lateral cerebral ventricle. When EGF and fibroblast growth factor 2 were injected into the lateral cerebral ventricles of rats, excitatory nerve stem cell hyperplasia increased 5–7-fold, accompanied by prolonged life spans. EGF-dependent lateral ventricular neurogenesis and hyperplasia are mediated by the EGF/EGFR signaling pathway[24]. EGF can induce proliferating neural precursor cells in the lateral cerebral ventricle to migrate to the surrounding brain tissue (including corpus striatum), but only a few cells differentiate into neuron-like cells[25]. Other reports of neurogenic differentiation also demonstrated that ADSCs exhibit a neuron-like morphology and express several proteins and genes consistent with the neuronal phenotype[2627282930].

ADSCs for the treatment of ischemic cerebrovascular disease

Cerebrovascular disease is a common ailment that is responsible for a very high number of deaths. Cerebrovascular disease can be divided into ischemic stroke and hemorrhagic stroke according to the pathological changes, and the former occurs at a high frequency. At present, ADSCs have been applied in orthopedics. Clinical research has confirmed that ADSCs possess many advantages, including their ease of procurement, wide availability, rapid expansion, low tumorigenesis, low immunogenicity, and absence of ethical constraints. Thus, ADSCs provide a new approach for the treatment of cerebrovascular disease.

Stereotaxic heterogenic transplantation of ADSCs for the treatment of cerebrovascular disease

Studies have demonstrated that transplantation of ADSCs can induce endogenous neural stem cells to proliferate, differentiate and migrate to improve the recovery of neural functions in rats with cerebrovascular disease (Table 1).

Table 1

Studies on stereotaxic heterogenic transplantation of adipose-derived stem cells (ADSCs) for the treatment of in ischemic cerebrovascular disease

Transplantation of ADSCs-conditioned medium (CM) for the treatment of cerebrovascular disease

The use of stem cell CM instead of direct implantation of stem cells is a feasible approach to overcoming the limitations of current cell-based therapy. Many cytokines and growth factors such as granulocyte-macrophage colony-stimulating factor, vascular EGF, hepatocyte growth factor, basic FGF (bFGF), transforming growth factor and insulin-like growth factor-1 have been identified in the CM of various stem cells[3940414243444546], which may be responsible for the paracrine protective effects of stem cells against various cytotoxic insults. Previous studies have reported the use of stem cell CM for experimental regenerative therapies. For example, CM obtained from amniotic fluid-derived MSCs and ADSCs significantly enhanced wound healing[47]. Endothelial progenitor cell CM induced neovascularization in a rat hindlimb ischemia model[48]. Cho et al[49] reported that continuous infusion of CM or aMEM medium (0.5 L/h) into the lateral ventricle that was initiated 8 days after surgery and maintained for 7 days induced significant functional and structural recovery after stroke as a consequence of enhanced neovascularization, reduced neural cell apoptosis, and milder astrogliosis. In Egashira's study[50], intracerebroventricular administration of 30- and 100-fold concentrated murine ASC-CM 1 hour prior to middle cerebral artery occlusion (MCAO) resulted in a dose-dependent reduction in the infarct volume and the brain swelling. The administration of murine ASC-CM immediately after MCAO was also effective, but administration 2 hours after MCAO was not. Pretreatment with 100-fold concentrated murine ASCCM at 10% of the total culture volume significantly reduced glutamate-induced excitotoxicity in SH-SY5Y cells. A similar reduction in the MCAO-induced infarction volume was seen following administration of 100-fold concentrated human ASC-CM or murine ASC-CM. These results demonstrated that ASC-CM appears to promote recovery after experimental ischemic stroke in both in vivo and in vitro models. These findings suggest the feasibility of ASC-CM administration as a therapy for acute stage stroke.

Mechanisms of action of transplanted ADSCs in the treatment of cerebrovascular disease

A deeper understanding of the molecular mechanisms underlying the differentiation of ADSCs would facilitate the application of ADSC transplantation in the treatment of cerebrovascular disease. miRNAs can regulate gene expression by inhibiting mRNA translation or promoting mRNA degradation. Previous studies have shown that miRNAs are highly correlated with stem cell self-renewal and differentiation, and play important roles in regulating stem cell activity[5152]. miRNA-125 has been described as a key regulatory molecule of the bioactivities of neural stem cells[53], and several miRNAs have been shown to maintain the stem cell phenotype and induce directional differentiation[54]. For example, miR-138 expression is downregulated during differentiation of ADSCs into adipocytes, and inhibition of EP300-interacting inhibitor of differentiation 1 can suppress this differentiation[55]. Furthermore, high miR-184 expression can promote the proliferation of neural stem cells and inhibit their differentiation[56].

The following four routes may describe the underlying mechanisms of action of ADSCs in the treatment of cerebrovascular disease. (1) ADSCs substitute for absent neural cells via differentiation into neurons and glial cells; (2) ADSCs promote neovascularization and improve focal blood supply by secreting various vascular growth factors and differentiating into vascular endothelial cells; (3) ADSCs facilitate functional recovery by secreting neurotrophic factors; and (4) ADSCs promote self-repair by stimulating endogenous neural stem cells to differentiate and mature.

CONCLUSION

ADSCs have many potential advantages for clinical applications. However, there are still many problems to be solved to enable the clinical application of ADSCs. Various factors affect the differentiation of ADSCs into nerve cells and the mechanisms are not fully understood. Additionally, it is still not clear whether the induced neuron-like cells or glial-like cells exhibit nerve cell functions. Further nerve electrophysiology and neural biochemical experiments are needed to confirm this. At present, the biological activities of ADSCs are well-characterized in vitro, but their regulation by various factors in body fluids in vivo is unclear. Future research should focus on: (1) in vivo experiments, changes in gene expression that induce differentiation, and ensuring the stability of recombinant genes in expanded cell populations; (2) avoiding immunological rejection in clinical applications and immune disorders after transplantation in vivo; (3) excluding infectivity and the potential for carcinogenesis. It is currently unknown whether stem cell transplantation can improve function in stroke patients by replacing the damaged cells and reconstructing the neural circuits. Therefore, the key issues to be solved regarding stem cell transplantation for the treatment of stroke are how to improve the differentiation of transplanted stem cells into specific neurons and glial cells, and their integration into host brain structure and function.

Table 2

Studies on heterogenic intravenous transplantation of human adipose-derived stem cells (hADSCs) for the treatment of cerebrovascular disease

Table 3

Studies on allogeneic intravenous transplantation of adipose-derived mesenchymal stem cells (ADSCs) for the treatment of cerebrovascular disease