Fig. 4.1
Summary of induction of iPSCs, iNSCs, and iNCs and direct in vivo reprogramming (Modified from Yamashita et al. [28]). (a) Overexpression of Oct3/4, Sox2, Klf4, and c-Myc can convert somatic cells such as skin fibroblasts into iPSCs. Neuronal cells can be obtained after differentiation in the cell culture system. (b) Overexpression of Sox2 with other factors can convert skin fibroblasts into iNSCs. Both neuronal and glial lineages can be obtained from iNSCs. (c) The combination of Asc1, Brn2, and Myt1l with other factors can directly convert skin fibroblasts into iNCs (direct reprogramming methods). (d) Overexpression of NeuroD1 with other factors can convert endogenous glial cells into neuronal cells in vivo (in vivo direct reprogramming methods)
In this chapter, we briefly review recent progress of cell transplantation/replacement therapy with iPSCs/iNSCs/iNCs alongside our recent findings.
4.2 Therapeutic Effect of Transplantation of Human IPS Cells in an Animal Model
In 2006, Prof. Yamanaka first established murine iPSCs by overexpressing four transcriptional factors (Oct3/4, Sox2, c-Myc, and Klf4) in mouse fibroblasts. Of note, they found that these key transcription factors (TFs) from 20 candidates were strongly expressed in embryonic stem cells (ESCs) [1]. iPSCs can retain high replication competence and pluripotency and can differentiate into various kinds of cells, similar to ESCs, indicating that overexpression of key TFs can change cell fate. Since iPSCs can be produced from a patient’s skin fibroblasts, there are no immunoreactive and/or ethical issues associated with ESCs. Therefore, iPSCs are believed to be a promising cell resource for cell transplantation/replacement therapy. Several scientific papers have demonstrated that human iPS-derived neuronal stem cells/neuronal progenitors, when transplanted into the stroke murine model brain, showed a therapeutic effect such as the recovery of motor function (Table 4.1). Notably, Oki et al. generated long-term self-renewing neuroepithelial-like stem cells from adult human fibroblast-derived iPSCs and transplanted them into the stroke mouse model. They found that motor function had already recovered by the first week after transplantation. They also confirmed that transplanted cells survived without forming tumors for at least 4 months. In their experiment, functional recovery was observed soon after cell transplantation, and the observed therapeutic effect was regarded to be derived from a neurotrophic effect caused by the release of transplanted cells [2].
Table 4.1
Therapeutic effect of transplantation of iPS-derived neuronal cells in the ischemic stroke model
Original cells | Induced cells | Main findings | References |
|---|---|---|---|
Human skin fibroblasts | Neuroepithelial-like stem cells | iPS-derived neuroepithelial-like stem cells were transplanted into poststroke striatum of MCAO mice 1 week after the induction of cerebral ischemia. Motor functional recovery was observed 1 week after cell transplantation. Authors found that part of transplanted cells survived for at least 4 months, showing that grafted cells exhibited electrophysiological properties of mature neurons and received synaptic input from host neurons | Oki et al. [2] |
Human skin fibroblasts | Neuronal progenitor cells | iPS-derived neuronal progenitor cells were transplanted into poststroke striatum of MCAO mice 1 week after the induction of cerebral ischemia. Motor functional recovery was observed 6 weeks after cell transplantation. At this time, part of the grafted cells survived, expressing some neuronal markers | Gomi et al. [15] |
Human skin fibroblasts | Neuroepithelial-like stem cells | iPS-derived neuroepithelial-like stem cells were transplanted into the poststroke cortex of MCAO rats 48 h after the induction of cerebral ischemia. Motor functional recovery was observed 5 months after cell transplantation. Authors confirmed that grafted cells exhibited electrophysiological properties of mature neurons and received synaptic input from host neurons | Tornero et al. [16] |
Human skin fibroblasts | Neuronal progenitor cells | iPS-derived neuronal progenitor cells were transplanted into the poststroke striatum of MCAO mice 1 week after the induction of cerebral ischemia. Motor functional recovery was observed 2–3 weeks after cell transplantation. Authors found that part of transplanted cells survived at least for 1 month, showing that grafted cells express neuronal markers such as NeuN. At 6 and 12 months after cell transplantation, tumor formation was not detected | Mohamad et al. [17] |
4.3 Discovery of iN Cells
Some Japanese research groups have started or plan to conduct clinical transplantation therapy trials using iPS cells for age-related macular degeneration, spinal cord injury, and Parkinson disease [3]. However, iPS cells can form tumors, especially in pathological conditions such as poststroke [4]. In addition, it is likely to be difficult to monitor tumor formation for more than 2 years, even if iPS cells are transplanted into a mouse model. Therefore, a new technology and strategy to induce neuronal cells in damaged brains is required. Research findings using iPS suggest that master TFs regulating the overexpression of ES cells could convert fibroblasts to ES cell-like iPS cells. From this finding, many researchers have overexpressed neuron-specific TFs in skin/lung fibroblasts and tried to convert these fibroblasts into neuronal cells. In 2010, Wernig et al. first established murine-induced neuronal cells (iNCs) by introducing three neuron-specific TFs (Ascl1, Brn2, and Myt1l) into mouse fibroblasts. They found that these iNCs showed a glutamatergic neuronal phenotype with synapses and action potential, as recorded by electric patch-clump analysis [5]. Various kinds of iNCs, including dopaminergic neurons and motor neurons, have been reported (Table 4.2). Interestingly, Ascl1 appears to be a key factor in the induction of iN cells, and the specific combination of Ascl1 plus other factors can convert somatic cells to specific neuronal cells. In cell transplantation therapy, it has already been reported that induced dopaminergic neurons showed a therapeutic effect against 6-hydroxydopamine (6-OHDA)-treated rats by attenuating the level of striatal dopamine [6]. iNCs can be produced without passing through the multipotent stem cell linage as iPS cells can be regarded as safer and easier to induce within a relatively short time frame, compared with iPS cells. However, the cell cycle of iN cells stops during cell conversion, making it difficult to prepare sufficient quantities of iNCs for cell transplantation therapy. To overcome this problem, induced neuronal stem cells (iNSCs) were developed. In 2012, Han et al. demonstrated that a combination of TFs (Sox2, Brn4, Klf4, c-Myc) successfully induced mouse fibroblasts directly to iNSCs [7]. Han and collaborators evaluated the therapeutic effect of cell transplantation using iNSCs in the spinal cord injury rat model. They found that engrafted iNSCs could differentiate into neuronal lineages forming synapses and enhancing the recovery of locomotor function [8]. iNSCs can thus be regarded as a promising cell resource for cell transplantation/replacement therapy.
Table 4.2
Scientific reports showing direct reprogramming from fibroblasts to neuronal cells
Target cells | Original cells | Combination of transcriptional factors for reprogramming | References |
Glutamatergic neurons | Mice fibroblasts | Ascl1, Brn2, Myt1 | Vierbuchen et al. [5] |
Mice hepatocytes | Ascl1, Brn2, Myt1 | Marro et al. [18] | |
Human fibroblasts | Ascl1, Brn2, Myt1, NeuroD | Pang et al. [19] | |
Human fibroblasts | Ascl1, Brn2, Myt1, Olig2, Zic1 | Qiang et al. [20] | |
Human fibroblasts | Ascl1, Myt1, NeuroD2, miR-9/9* and miR-124 | Yoo et al. [21] | |
Human fibroblasts | Brn2, Myt1, miR-124 | Ambasudhan et al. [22] | |
Dopaminergic neurons | Mice/human fibroblasts | Ascl1, Lmx1a, Nurr1 | Caiazzo et al. [23] |
Mice fibroblasts | Ascl1, Lmx1a, Nurr1, Pitx3, Foxa2, EN1 | Kim et al. [6]
Related posts: Cell Therapy for Ischemic Stroke with Bone Marrow Stromal Cells
Role of Biomaterials as Scaffolding in Cell Therapy for Stroke
Review of Previous Clinical Trials and Guidelines of Cell Therapy
 Neural Stem Cells/Neuronal Progenitor Cells
 In Vivo Cell Tracking Techniques for Applications in Central Nervous System Disorders
 Mesenchymal Stem Cells
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