A major discrepancy in the results of preclinical studies is whether graft cells have the ability to migrate to a cerebral lesion, although mechanisms of MSC transmigration across the blood–brain barrier (BBB) have been analyzed (Liu et al., 2013). The accumulation of graft cells in the lesion is expected to directly enhance neuroprotection and cell replacement in infarcted tissue. A comparison of different administration routes revealed that transarterial delivery was more successful in order to detect graft cells in the brain than transvenous delivery, although several studies reported a decrease in the number of detected cells in the later phase (Ishizaka et al., 2013; Mitkari et al., 2013). The transvenous route induced fewer side effects than intra-arterial infusion; however, physiologically, graft cells must pass through several traps, such as the lung and BBB. Although, the BBB can be disrupted by ischemic insult around the damaged areas, MSCs may have the basic ability to transmigrate the BBB as immune cells in response to homing signals to the lesion (Liu et al., 2013). Nonetheless, there are certainly successful examples demonstrating the integration of graft cells in the peri-infarct area even after transvenous infusion from a peripheral vessel (Table 1).

Classically, immunohistological analysis is a standard method to detect MSC migration, but recent imaging techniques, such as magnetic resonance imaging (MRI) with magnetic cell labeling (Detante et al., 2012; Canazza et al., 2013) and nuclear imaging using ^99mTc-labeled graft (Detante et al., 2009; Vasconcelos-Dos-Santos et al., 2012), have been proposed to reveal the distribution of MSCs. Subsequently, a phase I clinical trial employing ^99mTc – single photon emission computed tomography (SPECT) for assessment of biodistribution of the labeled grafts in subacute patients have safely conducted (Rosado-De-Castro et al., 2013b). The findings of these recent analytical methods may resolve the question of accurate distribution of graft cells.

Many preclinical studies have also reported differences in infarct volume reduction and functional recovery (Hao et al., 2014). Assessment methods of functional recovery vary, although there certainly are popular tests in animal studies, such as the treadmill test or Roger’s test. Therefore, differences in functional assessment may simply be based on differences in the employed assessment methods. On the other hand, it is more difficult to elucidate discrepancies in infarct volume reduction. In vivo studies with rodents have been conducted to investigate the changes in infarct volume reduction by direct measurement of the brain tissue after decapitation. Regarding clinical applications, non-invasive methods, such as MRI, may be beneficial to translate the findings of in vivo studies to clinical settings. Although the availability of mechanical devices varies among laboratories, the development of alternative clinical methods is recommended for future in vivo experiments.

Another problem is whether MSCs isolated from different tissues also differ. MSCs are obtained from diverse mesodermal tissues, i.e., bone marrow, adipose tissue, dental pulp, or cord blood. MSCs from different sources show different characteristics in vitro (Kern et al., 2006; Hsiao et al., 2012). Therefore, comparative study for different cell sources as conducted by Gutierrez-Fernandez’s group is important, however, the therapeutic effects in similar experimental ischemic stroke models also differ in transvenous administration studies (Ikegame et al., 2011; Steiner et al., 2012; Gutierrez-Fernandez et al., 2013) compared to intra-arterial administration studies that have shown graft cells in the lesion. (Table 1)

On the other hand, nuclear imaging is another available method to assess the therapeutic effectiveness. Diffusion and perfusion-weighted imaging provide information of blood supply in the brain (Canazza et al., 2013). Furthermore, functional MRI is employed by experimental studies in rodents, which unable to assess functional recovery (Suzuki et al., 2013) and even neural network by analyses of resting state functional MRI (Canazza et al., 2013). The neural integrity has been investigated by ¹²³I – Iomazenil SPECT (Saito et al., 2013). A ¹⁸F-FDG positron emission tomography study have measured glucose metabolism after MSC therapy in rats for cerebral ischemia (Miyamoto et al., 2013). For assessment of functional recovery, these methods from more bio-functional aspect would be practical in addition to observations of behavioral change.

The direction of differentiation also remains controversial for in vivo experimental studies. Although MSCs are derived from mesenchymal tissue, they exhibit multipotency and transdifferentiation into ectodermal lineages, including neural cells, both in vitro and in vivo (Zuk, 2013). Previous in vitro immunochemistry studies have demonstrated the ability of MSCs to differentiate into cell types that comprise the neurovascular unit, including neurons, astrocytes (Wislet-Gendebien et al., 2004), and endothelial cells (Hess et al., 2002; Planat-Benard et al., 2004). Moreover, possible differentiation abilities toward oligodendrocyte lineage (NG2-positive cells; Shen et al., 2006), specific types of neurons, such as glutamatergic neurons (Yu et al., 2014), and smooth muscle cells of vessels (Kubis et al., 2007) have been demonstrated. In vivo studies have reported that graft cells detected in the lesion result from neuronal or glial differentiation (Guzman et al., 2008). However, one study demonstrated the vascular fate rather than differentiation to neural lineages (Kubis et al., 2007).

To ensure the practical differentiation, in addition to these morphological, immunohistochemical, or genetic assessments, cells should be further examined. With respect to neural differentiation, neurotransmitter responsiveness or electrophysiological recording is required to examine their function as a neuron (Yang et al., 2011). Moreover, when MSCs are employed, absence of cell fusion also should be excluded. Though the MSC’s rate of spontaneous cell fusion is only 2–11 clones per million cells (Terada et al., 2002), and the mechanism may also participate in the tissue repair, nonetheless, biologically it should be distinguished from differentiation. BMSC and ASC are observed the neural differentiation that can show neural function in earlier studies. First, Ashjian et al. (2003) recorded K⁺ current on neuronal cells induced from ASC. Cho et al. (2005) reported synaptic transmission, and Wislet-Gendebien et al. (2005) showed action potential of the neuron-like cells differentiated from BMSC.

With the exception of the acute phase after ischemic insult, both allogenic and autologous grafting of MSCs can be prepared. Although the efficacy of technologies has improved, besides the advantage of MSCs in immunomodulation, theoretically allogenic grafts cannot ameliorate all concerns regarding transinfection or immunological side effects. Autologous grafts can overcome the problems related to allogenic cells. Nonetheless, at the present stage, other than obtaining the major MSCs, the use of both BMSCs and ASCs requires invasive procedures. Bone marrow aspiration and harvesting of adipose tissue are considered safe and established techniques; however, because ischemic stroke patients usually take antiplatelet or anticoagulant agents, and in some case, the patient may be intolerant to other conditions, further less invasive methods, such as the use of peripheral blood, present alternative sources of cells. As mentioned in the previous section, each type of MSCs from different cell sources tend to exhibit original traits or abilities, although they meet the criteria of MSCs. Knowledge regarding defined factors/conditions for MSC-fate regulation could enable the preparation of homogenous MSCs, even from peripheral blood (Meng et al., 2013).

Autologous grafts may have an additional advantage over allogenic grafts. In preclinical observations, MSCs reportedly developed function following contact with a conditioned media (Egashira et al., 2013), serum (Honmou et al., 2011), or cerebrospinal fluid from patients (Orito et al., 2010), which is reflected in the biological responses to invasive stimulation. It is possible that MSCs may achieve proper function in reaction to insults (Kurozumi et al., 2005; Xin et al., 2013). Therefore, graft cells harvested from ischemic stroke patients may gain more favorable function than allogenic grafts from those who are not affected by ischemic insults. Strikingly, the first nonrandomized clinical trial for a protocol with autologous BMSCs and serum has been shown to be safe and effective (Bang et al., 2005; Lee et al., 2010; Honmou et al., 2011). A 5-year randomized trial also began in 2012, which will provide further information regarding autologous stem cell therapy (Kim et al., 2013).

From a pharmacological viewpoint, the actions of agents should be confirmed after administration. If MSCs are regarded as a type of biological drug, then differences in differentiation ability should be better clarified.

Emerging induced pluripotent stem cells (iPSC) studies have shown promising benefits in the field of regenerative medicine that could have at least two major impacts on MSC studies. These findings may be useful to settle the controversies listed above, particularly those regarding the direction of differentiation of graft cells in the host and differences in the characteristics of MSCs originating from the cell source.

First, the appearance of iPSCs indicates the potential of multipotency in somatic cells (Takahashi and Yamanaka, 2006), which is supported by observations of differentiation into either neural or endothelial cells in MSCs. Although many reports have demonstrated the ability MSCs of mesodermal origin to differentiate into other type of germ cells of ectodermal lineages (neural cells) and endodermal lineages (insulin-producing cells), which could indicate multipotency, the defined conditions for MSCs to differentiate into neural cells remain uncertain. In the infancy of stem cell research, cell fusion and contamination of neural crest cells were suggested as the mechanism of a graft cell to express neural markers in the host tissue after cell administration (Wrage et al., 2008; Maltman et al., 2011). If the postulates reveal to be the main mechanism, neural marker expression can’t be called neural differentiation, which unable MSC to be called “stem cell.” Therefore, until recently, the term “MSC” containing the term “stem cell” had its pros and cons, and thus, MSCs were called stromal cells. However, successful reprogramming of skin fibroblasts to the multipotent state has provided more information to support the multipotency of MSCs.

Second, induction techniques may contribute to further elucidate the quality control mechanisms for the use of MSCs. Protocols for chemical induction to neuron or glia had been developed recently (Safford and Rice, 2005; Franco Lambert et al., 2009; Yu et al., 2011). Following the publication of methods to harness and propagate iPSCs, other methods related to direct conversion from fibroblasts to neuronal cells by defined transcription factors have been reported (Vierbuchen et al., 2010; Yang et al., 2013). The neural lineage is composed of induced neuronal (iN) cells, induced neural progenitor cells (iNPCs), and induced NSCs (iNSCs; Yang et al., 2011; Abdullah et al., 2012; Corti et al., 2012; Shi and Jiao, 2012). Moreover, iPSC-derived MSCs (iPSC–MSCs) were identified (Jung et al., 2012). There are multiple pathways for neural induction. As listed in the Table 2, in addition to defined transcriptional factors for direct conversion, microRNA (Feng and Feng, 2011; Pham and Gallicano, 2012; Bian et al., 2013) or other epigenetic factors (Namihira and Nakashima, 2011) can contribute to differentiation. The definitive conditions to propagate/identify iN cells, iNSCs, iNPCs, or iPSC–MSCs may be useful to propose a standard protocol for the required type of MSCs.

Lancaster et al.’s (2013) team developed a three-dimensional brain tissue from iPSCs by the floating culture method. To obtain functional recovery in vivo, several groups have shown that tissue regeneration or replacement of damaged tissue with ex vivo materials is not always necessary (Table 1). Particularly in the brain tissue, repair of the neural circuitry is required to improve function. Nonetheless, tissue engineering using scaffolds (Mahmood et al., 2013) or novel organogenesis methods present possible transplantation treatments to recover neurological deficits.