Implantation of human umbilical cord mesenchymal stem cells for ischemic stroke: perspectives and challenges

Abstract

Ischemic stroke is a focal cerebral insult that often leads to many adverse neurological complications severely affecting the quality of life. The prevalence of stroke is increasing throughout the world, while the efficacy of current pharmacological therapies remains unclear. As a neuroregenerative therapy, the implantation of human umbilical cord mesenchymal stem cells (hUC-MSCs) has shown great possibility to restore function after stroke. This review article provides an update role of hUC-MSCs implantation in the treatment of ischemic stroke. With the unique “immunosuppressive and immunoprivilege” property, hUC-MSCs are advised to be an important candidate for allogeneic cell treatment. Nevertheless, most of the treatments are still at primary stage and not clinically feasible at the current time. Several uncertain problems, such as culture conditions, allograft rejection, and potential tumorigenicity, are the choke points in this cellular therapy. More preclinical researches and clinical studies are needed before hUC-MSCs implantation can be used as a routinely applied clinical therapy.

Keywords

human umbilical cord / mesenchymal stem cells / ischemic stroke / cellular therapy / transplantation

Introduction

Ischemic stroke is one of the leading causes of motor, sensory, cognitive dysfunctions and death all over the world. Unfortunately, except the short time window required thrombolytic therapy, to date no effective treatment is proved to promote neuronal function recovery.

In the latest decades, stem cell-based therapies have become a potential approach to restore function in stroke [ 1]. Human umbilical cord mesenchymal stem cell (hUC-MSC) is a unique, accessible, and non-controversial source of stem cell which can enhance neurogenesis and angiogenesis in animal models of ischemic stroke [ 2]. In this review, we collected data on mechanisms and strategies of MSCs-based treatment, and evaluated the potentials and challenges of hUC-MSCs transplantation for ischemic stroke.

Sources of stem cells

Various types of stem cells have been isolated from a variety of tissue including preimplantation embryos, fetuses, birth-associated tissue and adult organs. Based on biochemical and genomic markers, these cells can be broadly classified into embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), and hematopoietic stem cells (HPSCs).

ESCs are pluripotent stem cells which theoretically can differentiate into all cell types of human body. However, ESCs suffer from a series of constraints including ethical concerns, limited availability, potential for teratoma formation and immune rejection [ 3]. Induced pluripotent stem cell (iPSC) is an alternative to ESCs, and has been generated from many cell types including cells from umbilical cord (UC) [ 4]. Although the use of patient-specific iPSCs will overcome the immune rejection obstacle, a great deal of work is needed before these cells can be used as a routinely applied therapy. The risk of tumorigenesis is particular important when using iPSCs, which are characterized by the ability to form teratomas in animal models [ 5].

HPSCs have limited plasticity since they can only differentiate into blood and blood-related lineages. In addition, there are only a small number of HPSCs in bone marrow and umbilical cord (UC), the cells require ex vivo expansion for clinical application [ 6].

Fetal MSCs are controversial as they are derived from human abortuses. Since the group of Pittenger isolated the multipotent MSCs from bone marrow first, the bone marrow has become the primary source to obtain MSCs [ 6]. Bone marrow mesenchymal stem cells (BM-MSCs) are the most extensively investigated MSCs and are considered the “gold standard” cell source in regenerative medicine. However, there are some limitations in the applications of BM-MSCs, including the small quantity of MSCs in marrow, the significant decrease of proliferation and differentiation potential with age. Moreover, the invasive harvesting procedure may lead to complications and morbidity [ 7].

Extra-embryonic MSCs harvested from UC represent a special stem cell type that combines some pluripotent properties of adult MSCs. With the close ontogenetic relationship to ESCs, hUC-MSCs have an immunoprivilege characteristic and proliferate faster than adult MSCs [ 8]. In addition, these cells can be isolated and used without any ethical problems, since extra-embryonic tissue is normally discarded after birth.

Isolation and culture of hUC-MSCs

hUC-MSCs can be derived from perivascular tissue and Wharton’s jelly in UC [ 9]. Wharton’s jelly means the mucoid connective substance which surrounds and supports the blood vessels in the cord. In 2003, Romanov et al. isolated the cells expressing MSCs markers from human umbilical cord first. However, the isolation and culture of hUC-MSCs are still controversial now. There are mainly two techniques: explant culture and enzymatic digestion, while the first way is more popular. In explant culture, which is also called “plate and wait,” the cord segments are simply plated in the medium to wait for the MSCs to migrate out [ 10]. Enzymatic digestion method means digesting Wharton’s jelly in an enzyme solution to release and obtain MSCs. The former technique is simple, effective, inexpensive and less labor-intense, but it is also time-consuming. The latter is more efficient when compared with explant culture, but overdigestion of tissue may result in diminished cellular viability, and altered cellular function [ 11]. There is a modified enzymatic digestion method which is more productive and economical: in the first step, the whole cord was digested to migrate vascular endothelial cells out, and then the rest of the cord was digested again to isolate the MSCs [ 12]. Besides, Dong et al. reported that the combination of collagenase type II, collagenase type IV, hyaluronidase, trypsin and DNAase could significantly improve the yield, viability and proliferation ability of MSCs, and the time of isolation could be shortened [ 13]. Liu et al. suggested that the implantation efficiency of hUC-MSCs could be increased when the cells were incubated in human umbilical cord serum, and the possible mechanism may be the reduction of senescent process through upregulating the heat shock protein 27 (Hsp27) [ 14].

Biological characteristics of hUC-MSCs

Surface markers

According to the statement by Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy in 2006, the characteristics of human MSCs should be defined in three aspects: in vitro growth pattern, the expression of specific surface antigens (positive for CD105, CD73 and CD90, and negative for CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR surface molecules), and the multipotent differentiation potential [ 15]. In the reported studies about hUC-MSCs, different reproducible expressions of cell surface epitope markers have been demonstrated by flow cytometric analysis. There are mainly four categories of surface markers which are shown in Table 1 [ 16– 21]. Nevertheless, the defining immunological markers of hUC-MSCs are still inconsistent among investigators. A standardized method should be identified to assess the implantation and immunomodulatory function of hUC-MSCs quickly and efficiently [ 22].

Immune properties

MSCs have two peculiar and outstanding immunological characteristics: immunosuppression and immunoprivilege [ 23, 24]. They can secrete some soluble factors (such as IDO, sHLA-G5) to suppress the function of lymphocytes and natural killer (NK) cells. By inhibiting the dendritic cells, they can induce regulatory T cells in central nervous system inflammatory diseases [ 25]. Furthermore, MSCs are capable of changing the secretion of dendritic cells, T cells, and NK cells, resulting in the feature of anti-inflammatory or tolerant phenotype [ 26]. There is in vitro evidence that MSCs can prevent the antigen-presenting cells from maturing and migrating, and they can change the expression of receptors that are important for antigen capture and processing [ 16, 27]. These studies have presented an attractive cellular therapy for ischemic stroke, and hUC-MSCs have shown great potential since they are easily and non-invasively available without ethical constraints [ 28, 29].

Except for the ability to modulate allogeneic immune system, hUC-MSCs do not appear to form teratomas, since they can express low levels of pluripotent embryonic stem cell markers (such as POUF1, NANOG, SOX2 and LIN28) [ 30– 33]. Meanwhile, hUC-MSCs could upregulate several cytokines (such as IL12A) which are associated with the induction of apoptosis, bringing on their anti-cancer feature [ 34]. Compared with other kinds of MSCs, hUC-MSCs are found to show increased expression of genes associated with cell chemotaxis and apoptosis. In addition, the immunomodulatory effects of hUC-MSCs did not change in response to neural induction [ 16]. This activity may play a key role in the rehabilitation of nervous system injuries.

Experimental studies about hUC-MSCs implantation in ischemic stroke

A number of researchers have reported the benefits of hUC-MSCs implantation in cerebral ischemia experiments. The animal model commonly applied is the transient rodent middle cerebral artery occlusion (MCAO) model. In the surgery, the right common carotid artery, external carotid artery and internal carotid artery were exposed. A nylon suture, with its tip rounded by heating near a flame, was advanced from the external carotid artery into the lumen of the internal carotid artery until the origin of the middle cerebral artery was blocked. Several hours later, animals were reanesthetized with isoflurane and blood reperfusion was achieved by withdrawal of the suture [ 35]. The benefits of neuroprotection were afforded by intravenous administration or intracerebral transplantation of hUC-MSCs, and the time lapse between injury and transplantation was different in the studies.

Reported by Liao et al., the rats were subjected to 2-h MCAO and received intracerebral transplantation of hUC-MSCs 24 h after surgery [ 36]. The transplanted cells could survive for at least 5 weeks in the brain, and this treatment could significantly reduce the injury volume of brain and neurological functional deficits. In addition, the therapy could substantially increase the vascular density and vascular endothelial growth factor (VEGF) expression in the cerebral hemisphere of infarction.

Similarly, the effectiveness of intravenous administration of hUC-MSCs was tested in rodent MCAO models in a time-of-administration study (time after stroke: day 1, day 7, day 30, and day 90) [ 1]. Marked functional recovery was observed when treatment was initiated at day 1, 7 or 30 but not at day 90 after stroke. The treatment could also enhance synaptogenesis, vessel density and the proliferation of SVZ cells, and apoptosis in the ischemic area was reduced. Lin et al. [ 37] found that rats receiving hUC-MSCs by intracerebral administration showed significant improvements in motor function, metabolic activity of cortical neurons and revascularization in the infarct cortex. The implanted cells survived in brain for at least 36 days and released neuroprotective and growth-associated cytokines, including brain-derived neurotrophic factor, platelet-derived growth factor-AA, basic fibroblast growth factor, angiopoietin-2, CXCL-16, neutrophil-activating protein-2, and VEGF receptor-3.

Potential therapeutic mechanisms

Replacement of damaged nervous cells

Prior study has indicated that neuronal replacement might be the possible mechanism of cell therapy in Parkinson’s disease [ 38]. There is evidence to show that the transplanted ESCs-derived neural stem cells could survive and differentiate into functional neurons, contributing to behavioral improvements and function restoration in experimental stroke models [ 39]. However, it may not be realistic that MSCs improve the neurological function of patients through a neuronal replacement mechanism [ 40]. Intracerebral transplanted MSCs derived from human umbilical cord blood could migrate to hippocampus and alleviate the ischemic brain injury in rats, but only the differentiation of MSCs to astrocytes was observed by histochemical study [ 41].

Proved by experiments, hUC-MSCs can differentiate into neural progenitors or neuron-like cells with the expression of neuron-specific markers such as nestin, Musashi-1, glial fibrillary acidic protein (GFAP), β-tubulin III, neuron specific protein TH, and neuron specific enolase (NSE) [ 42, 43]. In an in vivo study [ 44], intracerebral implantation of hUC-MSCs improved neurobehavioral function and reduced the infarct volume in rats with ischemic stroke. Three weeks after implantation, most of the implanted hUC-MSCs were present in the damaged hemisphere, and some of these cells expressed detectable levels of neuron-specific markers. However, functional active neuronal signal pathways were not detected by electrophysiological examination, indicating that the benefits of cell transplantation might be related to the neuroprotective effects rather than the formation of a new network between host neurons and the implanted cells.

It is demonstrated that hUC-MSCs could induce the immune response of host body under inflammatory conditions elicited by stroke, and the transplanted cells could be scavenged by the host immune cells. Weiss et al. [ 45] showed that hUC-MSCs were discovered 2 days after transplantation while no positive-staining hUC-MSCs were found 6 weeks later. The result indicated that the graft cells were cleared by the host immune system, the low-density graft was not able to support itself and the cells could not survive for a long time. Zhang et al. [ 1] reported that few hUC-MSCs were detected 2 months after intravenous transplantation in rat MCAO models, and they suggested that the implanted cells were possibly be eliminated by host immune system. Therefore, although hUC-MSCs have potentials to differentiate into neurons, neural differentiation and integration are unlikely to be the main mechanism of therapeutical function in cell therapy for stroke.

Promotion of endogenous neural cells

In the adult’s brain, the subventricular zone (SVZ) of lateral ventricle contains self-renewing multi-potent neural progenitors that are able to generate neurons, astrocytes and oligodentrocytes. Under normal conditions, glial fibrillary acidic protein (GFAP)-expressing cells in the SVZ include a neurogenic cell population that gives rise to olfactory bulb neurons only. Prior researches indicated that stroke increased neurogenesis in the SVZ, and newly generated neurons started to migrate toward ischemic boundary regions soon after stroke attacking. The generation of new neurons involves proliferation of progenitors, subsequent withdrawal from the cell cycle and differentiation [ 46]. After stroke, neural progenitor cells can proliferate and generate neuroblasts, migrate to the damaged area, become integrated, and differentiate to mature and functional neurons [ 45, 47, 48]. On the other hand, Jin et al. reported that suppressed SVZ neurogenesis led to worse functional outcome after experimental stroke in mice [ 49]. Raber et al. suggested irradiation in gerbils attenuated neurogenesis and exacerbated deficits of behavioral performance after cerebral global ischemia [ 50]. SVZ is the main source of new generated neurons, and the recovery after stroke may possibly be induced by SVZ progenitor proliferation and neurogenesis [ 51].

Besides, it is also reported that large amount of new born neurons eventually underwent cell death in stroke, while the transplanted MSCs could enhance proliferation of endogenous neurogenesis by suppressing apoptosis of newly generated cells [ 52]. Studies indicated that therapeutic effects of MSCs were at least partly ascribed to enhancing endogenous neurogenesis and protecting newborn cells from deleterious environment. MSCs were reported to promote the proliferation of neural stem cells or progenitor cells in SVZ, increase the expression of genes related to neural differentiation and protect the differentiated neurons [ 52, 53]. In the animal models of MCAO, intravenous transplantation of hUC-MSCs enhanced generation and neuronal differentiation of intrinsic cells in the brain, and made improvements on functional outcomes [ 1]. Taken together, stroke-induced neurogenesis and neuronal differentiation play important roles in brain repair and recovery, and the effects could be enhanced by hUC-MSCs transplantation.

Secretion of neurotrophic factors

The expression of neuroprotective and growth-associated factors were identified in MSCs, which contributed to the protective effect in tissue repair [ 54]. The implanted MSCs could improve the survival of host cells and promote regeneration of endogenous cells by factors excretion [ 55]. In prior researches, human MSCs were known to secrete biologically active cytokines or factors including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), colony-stimulating fator (CSF), stem cell factor (SCF), basic fibroblast growth factor (bFGF), platelet-derived growth factor-AA (PDGF-AA), angiopoietin-2, neutrophil-activating protein-2 (NAP2), and vascular endothelial growth factor receptor-3 (VEGFR-3) [ 56– 58]. Furthermore, it was shown that the secretion of trophic factors by MSCs was enhanced under post-ischemic conditions [ 59].

Transplanted through the basilar artery to canine ischemic stroke models, MSCs were revealed to successfully integrate into host brains, reduce infarction volume and ameliorate neurobehavioral function, which are closely related to factors releasing [ 60]. In the in vivo studies, hUC-MSCs implantation were able to improve the internal environment of the injured brain, enhance the survival of engrafted cells, and induce neurobehavioral improvement through the paracrine mechanism [ 35, 37]. In addition, the antiapoptotic signaling pathway was found to be activated by neurotrophic factors (such as BDNF), which induced reduction of cell death in brain tissue of stroke models [ 61]. Intravenous hUC-MSCs administration could also enhance angiogenesis, which was thought to be linked to the paracrine neurotrophic action of the implanted cells [ 62]. It could be concluded that neurotrophic factors released by MSCs are able to induce endogenous neuronal proliferation and differentiation, promote neovascularization and survival, decrease apoptosis, and bring on earlier recovery of neurological functions after brain injury [ 62, 63].

Induction of vascularization and angiogenesis

Suggested by prevenient researchers, vasculature plays a significant role in striatal neurogenesis after stroke. When stroke attacks, the vascular endothelial cells in brain start to proliferate, and more oxygen and nutrient were supplied to the ischemic tissue. During the next several months, neuroblasts gradually migrate toward the injured brain region with vascular remodeling and vessel density increasing. Therefore, the generation of new blood vessels is quite important for the survival, migration and differentiation of the closely located neuroblasts [ 64]. As a physiological repair process that naturally occurs in ischemic stroke, changes in vascularization can be promoted by MSCs transplantation. Existing evidence suggested MSCs could be induced to acquire the angiogenic and vasculogenic properties by producing appropriate cytokine milieu [ 65]. Through the promotion of vascular growth, MSCs could improve cerebral blood flow, and finally ameliorate the ischemic tissue damage [ 66].

On the other hand, the endothelial potential of hUC-MSCs has been confirmed in experiments. These cells were found to differentiate into vascular endothelial cells with additionally increasing expression of VEGF [ 67]. Moreover, Zhang et al. [ 1] found that hUC-MSCs were able to modulate the vascular system by increasing density of von Willebrand factor (vWF)-immunoreactive vessels. These studies seems to point toward the theory that administered hUC-MSCs induce angiogenesis and enhance brain blood supplying to reduce stroke-induced deficits, which is likely due to secreting paracrine factors.

Reduction of apoptosis

Neuron apoptosis have been detected in normal adult animal striatum by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL assay). Observations suggested that neuronal programmed elimination was a common feature in the adult mammalian brain, in which cells from neurogenic regions were largely eliminated through a self-renewal mechanism [ 68]. Previous study has demonstrated that stroke-induced cell apoptosis peaked at 1 to 2 days after stroke onset and last for at least 4 weeks, which significantly contributed to enormous death of newly generated cells (approximately 80% of the total) in ischemic brain injury [ 69].

Transplanted into rats with transient middle cerebral artery occlusion, MSCs could contribute to motor function improvement by reducing neuronal damage via inhibiting neuronal apoptosis [ 70]. Scheibe et al. reported that neuron apoptosis could be prevented and neuronal damage could be reduced by MSCs transplantation in oxygen-glucose deprivation (OGD) model of neurons [ 71]. hUC-MSCs treatment could promote endogenous neurorestoration and decrease apoptotic cell death till 2 months after stroke [ 1]. Additionally, it is proved that the capacity of MSCs to prevent apoptosis mainly depended on inactivating some proteolytic enzymes, such as metalloproteinases (MMPs) [ 72]. It is reasonable to speculate that, through an anti-apoptosis mechanism, MSCs can improve the survival of newly generated cells and repair the injured brain tissue after cerebral ischemia [ 54].

Prevention of inflammatory effect

Inflammation plays a critical part in ischemic stroke. When the stroke occurs, the brain will show a rapid and prolonged response of inflammatory process such as the activation of resident cells (mainly microglia), production of inflammatory factors and stimulation of various types of inflammatory cells (neutrophils, T cells, monocytes, and macrophages) [ 73]. All of these cellular events collaboratively contribute to cerebral injury eventually. Therefore, the inhibition of splenic function may be another mechanism of MSCs therapy for stroke [ 74].

It is reported that spleen adversely increased the blood-brain barrier permeability in rat MCAO models. This splenic response could be blocked by MSCs implantation, which suggested a novel immunomodulatory approach to mediate neuronal protection [ 75]. In the previous studies, MSCs were shown to interact with innate and adaptive immune cells and express immunomodulatory cytokines [ 76]. The transplanted MSCs could decrease the number of inflammatory cells released by spleen [ 77], upregulate anti-inflammatory cytokine IL-10 and downregulate proinflammatory cytokine IL-1β [ 78]. These reports supported the concept that hUC-MSCs participated in the early tissue remodeling process of cerebral infarction partly by controlling inflammatory response.

In summary, hUC-MSCs administration may aid in reducing the long-term impact of stroke via multiple mechanisms that still have to be fully elucidated [ 79]. Plenty of transplanted cells migrated to lung and liver, or became apoptotic eventually in host brain. Few originally delivered hUC-MSCs could retain residency and differentiate into active neural cells. Therefore, the indirect paracrine mechanisms, including the secretion of neurotrophic and anti-apoptotic factors, the anti-inflammatory ability, the capacity to induce neurogenesis and angiogenesis, are more likely to accelerate neurorestoration [ 80]. Potential therapeutic mechanisms of hUC-MSCs implantation for ischemic stroke are shown as Fig. 1.

The possible risks of the treatment

Despite the special biological characteristics and enormous potential benefits in application of hUC-MSCs, there are still some possible risks exist in hUC-MSCs based clinical therapy. One of the unresolved issues would be related to the application of allogeneic cells transplantation [ 81]. As previously discussed, hUC-MSCs can be used as a potential valid alternative to stroke, as they could prevent allograft rejection. However, the precise mechanism responsible for this effect and whether it will persist long-term in clinical treatment remain to be determined by further studies. In this case, allogeneic rather than autologous cells may expose the recipients to graft-versus-host diseases, which would complicate the treatment. Another potential risk is the inducement of infection [ 75]. Delivering MSCs to cerebrospinal fluid or ischemic cortex would likely elevate the probability of infection in comparison with intravenous implantation.

In addition, there are more and more evidences to show that MSCs have the potential to migrate into tumor and promote tumor cell proliferation, invasion and metastasis [ 82, 83]. Using an nude mice model of colon cancer, orthotopic transplantation of cancer cells mixed with MSCs resulted in greater tumor weight and significantly lower survival rate than delivery of KM12SM cells alone did [ 84]. The mechanisms may lie on suppressing the antitumor immune response, directly providing nourishment to tumor cells and inhibiting apoptosis of tumor cells [ 85, 86]. Although hUC-MSCs did not produce tumors in vitro experiments, further researches are necessary to evaluate their tumorigenic possibilities. And there should be more systematic reports about the complications in hUC-MSCs-based treatment to evaluate the benefit-to-risk ratio of cellular therapy in ischemic stroke.

Conclusions

Human umbilical cord mesenchymal stem cells are collected from Wharton’s jelly and perivascular region in the umbilical cord, which is generally considered to be a “leftover” after childbirth and can be easily gained without ethical controversy. hUC-MSCs have the unique property of self-renewing and proliferation, and no host response or tumorigenesis is induced by hUC-MSCs in animal experiments. Researchful evidence in preclinical models indicates that hUC-MSCs transplantation therapy is promising to achieve neural repair and protection.

Clinical trials on MSCs transplantation for stroke are currently ongoing. In patients with ischemic stroke, intravenous transplantation of autologous MSCs derived from BM could improve functional recovery without safety concerns for death, stroke recurrence, or serious adverse events up to 1 year [ 87]. Lee et al. reported a long-term follow-up study for 5 years and observed no evidence for venous thromboembolism, systemic malignancy or systemic infection in any of the patients following MSCs infusion [ 88]. In 2011, the first report of hUC-MSCs in a human clinical application is reported [ 89]. hUC-MSCs were found to have superior proliferative potential and more suppressive effects on peripheral blood mononuclear cell proliferation compared with bone marrow MSCs. However, currently there is no clinical trial which has been applied on hUC-MSCs transplantation for ischemic stroke (http://clinicaltrials.gov). It’s too early to decide that hUC-MSCs administration can be clinically developed to promote recovery after stroke. The animal model can not simulate all aspects of pathological process in stroke, and the behavior changes after cell transplantation in animal models may only partly reflect the situation in patients. Moreover, several uncertain problems, such as the culture conditions, inducement of allograft rejection and potential tumorigenicity, are the choke points in this cellular therapy. More preclinical researches and clinical studies are needed before these cells can be used as a routinely applied clinical treatment.

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