Direct Reprogrammed Neuronal Cells as a Novel Resource for Cell Transplantation Therapy

Introduction

The proportion of elderly individuals in the total population is rising in the industrialized nations of the world, resulting in an increase in the number of patients that suffer from neurological diseases, including Alzheimer's disease and ischemic stroke. For example, stroke is the second leading cause of death in the world and results in a drastic reduction in the quality of life (23). However, effective therapy for the chronic phase of stroke is not yet available because the reconstruction of a disrupted neuronal network is considered to be difficult.

As a novel strategy for neurological diseases, cell transplantation looks attractive because there is much scientific evidence showing the effectiveness of cell transplantation therapies in animal disease models and even in human patients (3,11). Embryonic stem cells (ESCs), which display natural self-renewal and multipotency, seem to be a promising cell resource. However, the serious ethical issue of destroying human embryos to obtain ESCs disturbs the application of ESCs for clinical treatment. Induced pluripotent stem cell (iPSC) technology is opening a new gate as a novel therapeutic strategy. Similar to iPSCs, direct reprogrammed cells, including induced neuronal cells (iNCs) and induced neural stem cells (iNSCs), have received attention as new cell resources. In this article, we focus on iPSCs, iNCs, and iNSCs and discuss the character, issues, and possibility of clinical application of cell replacement therapy for neurological diseases for each cell type.

iPSCs as Cell Resource for Cell Therapy

Murine iPSCs were first established by Yamanaka by introducing four transcriptional factors [v-myc avian myelocytomatosis viral oncogene homolog (c-Myc), octamer-binding transcription factor 3/4 (Oct3/4), sex-determining region Y box 2 (Sox2), and Krüppel-like factor 4 (Klf4)] into mouse fibroblasts (20). iPSCs are regarded as a promising cell source for cell transplantation therapy by supplying new neurons into a disrupted neuronal network because they can retain high replication competence and pluripotency and differentiate into various kinds of cells similar to ESCs. Since iPSCs can be produced from a patient's skin, they do not have the immunoreactive or ethical problems found for ESCs. Presently, Japanese research groups plan to perform clinical transplantation therapy trials of iPSC-derived pigment epithelium in age-related macular degeneration patients (14). However, as immature iPSCs have been reported to have high tumorigenic capacity (21), it now looks difficult to assure the safety of these trials. We reported that iPSC tumorigenesis can occur more easily in the post-stroke brain relative to a brain that has not suffered a stroke (25). In addition, in the murine model, it appears difficult to check tumor formation for longer than 2 years. Consequently, safety in human clinical trials has to be carefully considered. iPSCs that overexpress Yamanaka transcriptional factors can initialize cells (20). This important finding indicates the possibility that certain master transcriptional factors can change one cell fate to another. Therefore, many researchers have attempted to find other transcriptional factor combinations that would induce other differentiated cells to directly convert their cell fate.

Induced Neuronal Calls (iNCs)

Based on iPSC findings, many researchers hypothesize that overexpression of neuron-specific transcriptional factors may directly convert fibroblasts into neuronal cells. In 2010, murine iNCs were first established by Wernig by introducing three transcriptional factors [achaete-scute complex homolog 1 (Ascl1), brain-2 (Brn2 or OCT7), and myelin transcription factor 1-like (Myt1l)] into mouse fibroblasts. These iNCs were likely glutamatergic neurons with synapses that showed neuronal action potential in electric patch-clump analysis (24) (Fig. 1). Dopaminergic neurons and motor neurons have also been established with different combinations of transcriptional factors (Table 1). The first author of this article, Toru Yamashita, took part in a Columbia University project that successfully converted familial Alzheimer's disease patients' skin fibroblasts into glutamatergic iNCs, demonstrating that these patient-derived iNCs showed disease-specific cell characteristics, including amyloid precursor protein (APP) deposits and a high amyloid β 42 (Ab42)/Ab40 ratio (17). This report suggests that because iNCs can be produced within a relatively short period, 2–3 weeks, they can be a very powerful and convenient tool to study the mechanism of disease pathophysiology. In addition, as a cell resource for cell therapy, iNCs may be safer than iPSCs because iNCs can be produced without passing through the multipotent stem cell fate. It has been already reported that transplantation of induced dopaminergic neurons could attenuate 6-hydroxydopamine (6-OHDA)-treated rat behavior by improving the level of striatal dopamine (9). However, it may be not easy to prepare sufficient iNCs for cell transplantation because their cell cycle basically stops during cell conversion (24).

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Figure 1.

Induction of iPSCs, iNSCs, and iNCs. (a) Overexpression of octamer-binding transcription factor 3/4 (Oct3/4), sex-determining region Y box 2 (Sox2), Krüppel-like factor 4 (Klf4), and v-myc avian myelocytomatosis viral oncogene homolog (c-Myc) can convert skin fibroblasts into induced pluripotent stem cells (iPSCs). Neuronal cells can be differentiated from iPSCs in the cell culture system. (b) Overexpression of Sox2 with other factors can convert skin fibroblasts into induced neural stem cells (iNSCs). Both neuronal cells and glial cells can be obtained from iNSCs. (c) Overexpression of achaete-scute complex homolog 1 (Ascl1), brain-2 (Brn2 or OCT7), and myelin transcription factor 1-like (Myt1l) with other factors can directly convert skin fibroblasts into induced neuronal cells (iNCs) (direct reprogramming methods).

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Table 1.

Scientific Reports Showing Direct Reprogramming to Neuronal Cells

Target Cells	Original Cells	Combination of Transcriptional Factors for Reprogramming	Reference
Glutamatergic neurons	Mice fibroblasts	Ascl1, brn2, Myt1l	Vierbuchen et al. (24)
	Mice hepatocytes	Ascl1, brn2, Myt1l	Marro et al. (13)
	Human fibroblasts	Ascl1, brn2, Myt1l, NeuroD	Pang et al. (15)
	Human fibroblasts	Ascl1, brn2, Myt1l, Olig2, Zic1	Qiang et al. (17)
	Human fibroblasts	Ascl1, Myt1l, NeuroD2, miR-9/9*, and miR-124	Yoo et al. (26)
	Human fibroblasts	brn2, Myt1l, miR-124	Ambasudhan et al. (2)
Dopaminergic neurons	Mice/human fibroblasts	Ascl1, Lmx1α, Nurr1	Caiazzo et al. (4)
	Mice fibroblasts	Ascl1, Lmx1α, Nurr1, Pitx3, FoxA2, EN1	Kim et al. (9)
	Human fibroblasts	Ascl1, brn2, Myt1, Lmx1a, Fox2	Pfisterer et al. (16)
Motor neurons	Mice/human fibroblasts	Ascl1, brn2, Myt1l, NeuroD1, Lhx3, Hb9, Isl1, Ngn2	Son et al. (19)
Neural stem cells	Mice fibroblasts	Sox2, brn2, FoxG1	Lujan et al. (12)
	Mice fibroblasts	Sox2, brn4/Pou3f4, Klf4, c-Myc, E47/Tcf3	Han et al. (6)
	Mice/human fibroblasts	Sox2	Ring et al. (18)

Ascl1, Achaete-scute complex homolog 1; Brn2, brain-2; Myt1l, myelin transcription factor 1-like; NeuroD, neuronal differentiation; OLIG2, oligodendrocyte lineage transcription factor 2; Zic1, Zinc finger protein of the cerebellum 1; miR-9/9*, bifunctional microRNA strands 9; miR-124, microRNA 124; Lmx1α: LIM homeobox transcription factor 1, α; Nurr1, nuclear receptor related 1; Pitx3, paired-like homeodomain 3; Foxa2, forkhead box A2; EN1, engrailed homeobox 1; Lhx3, LIM homeobox 3; Hb9: homeobox 9; Isl1: islet 1 (ISL LIM homeobox 1); Ngn2, neurogenin 2; SOX-2, sex-determining region Y box 2; Klf4, Krüppel-like factor 4; c-Myc, v-myc avian myelocytomatosis viral oncogene homolog; E47/Tcf3, transcription factor 3.

iNSCs as Cell Resource for Cell Therapy

iPSCs tend to form tumors, and it is difficult to amplify them into iNCs. Thus, iNSCs are now regarded as one of the most promising cell resources. Many scientific groups already reported that ESC-derived NSCs/neural progenitors can partially recover the functional deficits in animal models of spinal cord injury (10), Parkinson's disease (5), and stroke (7). Moreover, it has been reported that Sox2, with or without other transcriptional factors, can directly induce iNSCs from fibroblasts or other cell types (6,12,18) (Fig. 1 and Table 1). Notably, Ring et al. showed that transplantation of iNSCs into mouse brains did not generate tumors within 6 weeks (18), suggesting that these cells have low tumorigenic potential, although longer periods of observation would be needed in the future. Furthermore, iNSCs have to be used and studied in animal disease models to know whether these cells have a therapeutic effect without tumor formation.

The Possibility of In Vivo Direct Conversion

Above, we discussed cell transplantation therapy with iPSCs/iNCs/iNSCs. Basically, in this strategy, some somatic cells, such as skin fibroblasts, have to be obtained from each patient and then converted to another cell fate in a culture system. In this strategy, viruses and other infectious materials can be transmitted to the transplanted cells, which will limit clinical applications. Thus, if endogenous parenchymal nonneuronal cells such as glial cells can be changed to a required neuronal cell fate, this could be a new straightforward route for the generation of new neurons. Addis et al. reported the direct reprogramming of astrocytes to dopaminergic neurons with Ascl1, LIM homeobox transcription factor 1, β (Lmx1β), and nuclear receptor related 1 (Nurr1) in a culture system (1). Human brain-derived pericytes also converted to neuronal cells in culture (8). Most recently, Torper et al. reported that endogenous mouse astrocytes could be directly converted into neuronal nuclei (NeuN)-positive neuronal cells in vivo (22). It is still unclear whether these in vivo direct reprogramming methods have sufficient therapeutic effects in animal disease models or human patients, although the above results indicate that in vivo direct reprogramming methods can be hopeful methods for clinical applications.

In this article, we briefly highlighted recent progress in the development of iPSCs, iNCs, and iNSCs for cell replacement treatment of damaged brains following neurological diseases. Even though the clinical application of iPSCs is ongoing, it is important to combine or choose appropriate strategies depending on the target disease.