Journal of Collaborative Healthcare and Translational Medicine

Role of the von Hippel-Lindau Tumor Suppressor Protein in Neuronal Differentiation of Somatic Stem Cells and its Application to Neuronal Regeneration: A Review

Review Article | Open Access

  • 1. Department of Neurosurgery, Yokohama City University, Japan
+ Show More - Show Less
Corresponding Authors
Hiroshi Kanno, Department of Neurosurgery, Yokohama City University, School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan,

von Hippel-Lindau tumor suppressor (VHL) protein functions to cause somatic stem cells to differentiate into neurons. Not only VHL protein but also a peptide derived from it shows this capability of eliciting neuronal differentiation by somatic stem cells. Up to now, rodent neural stem cells (NSCs) and human hair follicle stem cells, both of which are derived from ectoderm, have been shown to undergo neuronal differentiation triggered by VHL protein or a peptide derived from it. In addition, rodent skin-derived precursors and rodent bone marrow mesenchymal stem cells, both of which are derived from mesenchyme, also can differentiate into neurons by the same method. A 15-amino-acid peptide derived from VHL protein corresponds to the part of the sequence of VHL that binds to elongin C, which sequence is considered to be a domain for neuronal differentiation. The mechanism of neuronal differentiation of somatic stem cells by VHL is suggested to be inhibition of Stat 3. When VHL protein or oligopeptide derived from it was transferred into somatic stem cells and these cells were transplanted into the central nervous system of animals modeling a neuronal disease, the implanted cells differentiated into neuronal cells, resulting in recovery of neuronal functions. These facts suggest that somatic stem cells with transferred VHL protein or oligopeptide derived from it are candidates of donor cells for regeneration therapy of intractable neuronal diseases.


Kanno H, Higashida T, Kubo A (2014) Role of the von Hippel-Lindau Tumor Suppressor Protein in Neuronal Differentiation of Somatic Stem Cells and its Application to Neuronal Regeneration: A Review. J Transl Med Epidemiol 2(1): 1013.


•    von Hippel-Lindau tumor suppressor protein
•    Somatic stem cells
•    Neuronal differentiation
•    Peptide


von Hippel-Lindau disease is a hereditary multi-cancer syndrome that encompasses central nervous system hemangioblastoma, retinal angioma, renal cell cancer, and pheochromocytoma [1]. The causative gene composed of 3 exons and encoding 213 amino-acids, has been identified at chromosome 3p25 region [2]. The functions of this gene include inhibition of mRNA elongation [3], inhibition of hypoxia inducible factor (HIF)-1α [4], and induction of neuronal differentiation of neural progenitor cells [5]. These functions are normally controlled under normoxia, but not under hypoxia [4]. VHL protein is expressed in the cytoplasm of not only neural progenitor cells [5] but also in that of adult neurons [6]. The VHL protein is widely expressed in normal human tissues, but the cellular distribution of the protein is confined to the cytoplasm of specific cell types. High-level expression of the protein is observed in neural tissue, especially in Purkinje cells, Golgi type II cells, and cells in the dentate nucleus of the cerebellum, pontine nuclei, and inferior olivary nucleus of the medulla oblongata [6]. However, the significance of the expression of VHL protein in neurons has remained unclear. In addition, strong expression of VHL protein is observed particularly in the cytoplasm of renal tubule cells [6]. Since VHL protein plays an important role during neuronal differentiation [5], neuronal differentiation of somatic stem cells elicited by VHL protein might be useful for neuronal regeneration therapy by transplantation of somatic stem cells transfected with the VHL gene [5,7]. Here, in the context of translational research, we review the literature showing that VHL protein or a peptide derived from it promotes the differentiation of somatic stem cells into neuron-like cells [5,7], which ability suggests that these transfected stem cells might be useful as donor cells for neuronal regeneration therapy [8-12]. For use of somatic stem cells as donor cells for neuronal regenerative therapy, it is desirable that these cells differentiate into neuronal cells in the implant site. If naïve somatic stem cells are implanted into the central nervous system (CNS), they rarely survive; and those that do scarcely differentiate into neurons.

Somatic stem cells capable of neuronal differentiation are classified into ectoderm-derived stem cells and mesenchyme/ mesoderm- derived stem cells. Endoderm-derived stem cells scarcely show neuronal differentiation. Up to now, somatic stem cells reported to differentiate to neuronal cells when transfected with the VHL gene or a peptide derived from VHL protein are the following: rodent neuronal progenitor cells/neural stem cells (NPSs/NSCs) [6,9], human hair follicle stem cells (FSCs) [13], rodent skin-derived precursors (SKPs) [10,12], and rodent bone marrow mesenchymal stem cells (MSCs) [11]. The methods for isolation and culturing of these cells have been previously described [8,10,13,14].

Expression of VHL protein in neuronal progenitor cells

When neuronal progenitor cells (NPCs) were obtained from the forebrain of the E12 rat fetus, the expression levels of VHL protein and microtubule-associated protein (MAP)-2 were positively correlated during the development of the neuronal progenitor cells [6]. At day 1 after NPCs were placed in primary culture with bFGF, VHL protein and neuron-specific marker microtubule-associated protein (MAP)-2 were expressed at a low level, but by day 14 high levels of them were detected. On the other hand, when NPCs were cultured with EGF instead of bFGF, these cells expressed neither protein at a high level [6].

Neuronal differentiation of neuronal progenitor cells/neural stem cells transfected with the VHL gene

Through transfection of NSCs/NPCs with a VHL protein-expressing herpes virus vector or adenovirus vector, these cells differentiated into neuron-like cells in vitro and in vivo [5,7]. It was first reported that NPCs differentiated into neuronal marker-expressing cells through transfection with the VHL protein-expressing herpes simplex virus vector [5]. Significant expression levels of VHL protein and MAP-2 were observed at 3 and 24 hours, respectively, after transfection with the VHL gene; and the expression of both was observed significantly earlier than that in the control (P <0.0001) [6]. In addition, through transfection with a VHL protein-expressing adenovirus vector, NPCs differentiated into neuronal marker-positive cells [7]. On the other hand, the expression levels of astrocytic marker glial fibrillary acidic protein (GFAP) and oligodendroglial marker O4 decreased in NPCs following transfection with the VHL gene [7]. MAP-2 and tyrosine hydroxylase (TH) increased in expression in response to the transfection. The ratio of MAP-2 positive NPCs to total NPCs was significantly much higher in VHL gene-transfected NPCs treated with glial cell line-derived neurotrophic factor (GDNF) than in control NPCs without this factor (P<0.01) [7]. In line with these findings, it was also shown that a VHL mRNA antisense oligonucleotide inhibited the differentiation of NPCs [6].

Neuronal differentiation of somatic stem cells by intracellular delivery of the VHL peptide having the sequence of the elongin C-binding site

Through intracellular delivery of the 15 amino-acid peptide comprising the binding site for elongin C within the VHL protein (VHL peptide), the induction of neuronal differentiation of NPCs was examined. Elongin C, a ligand of the VHL protein, forms the elongin BC complex with elongin B, which complex then binds to elongin A, a transcription factor that elongates mRNA, or to BC-box proteins, which inhibit signal transcription factors such as Stat3, a transcription factor related to astrocytic differentiation [15]. Therefore, a peptide containing the binding site for elongin C was speculated to have the capability to induce neuronal differentiation owing to its neuronal differentiationinducing domain [8]. For the intracellular delivery of the VHL peptide, diloeoyl phosphatidyl ethanolamine lipid reagent for encapsulating molecules (BioPorter) was used. The BioPorterpeptide complexes become attached to the negatively charged cell surface and fuse with it, resulting in delivery of the captured protein into the cells. Four hours after the intracellular delivery, neurite outgrowth from NPCs was observed when the VHL peptide was used at a 0.3-µM concentration or above. Immunocytochemically, NPCs showed increased expression of MAP-2 after the delivery of the peptide, but decreased expression of GFAP [8]. In the electrophysiological study, voltage-gated inward and outward currents were recorded in the whole-cell patch-clamp configuration. In whole-cell recordings of the VHL peptide-containing NPCs, the depolarizing voltage steps elicit both large outward potassium currents and fast inward Na+ currents, which are hallmark features of differentiated neurons [8]. Furthermore, through the intracellular transfer of the protein transduction domain (PTD) linked to the VHL peptide (PTD-VHL peptide), NSCs differentiated into neuronal marker-positive neuron-like cells in vitro [9]. The majority of NSCs with transferred PTD-VHL peptide expressed neuron-specific markers such as neurofilament (NF)-H, MAP2, and Tuj-1, whereas the minority were positive for the stem cell marker nestin or the astrocytic marker GFAP. By western blotting for NSCs done 7 days after treatment with PTD-VHL peptide, a significantly greater amount of NF-H protein was observed in the PTD-VHL-peptide-bearing cells than in the PTD-peptide alone-treated group. In addition, a significantly greater amount of MAP2-protein was seen in the former than in the latter [9].

Similarly, the transfer of PTD-VHL peptide induced neuronal differentiation in skin-derived precursors (SKPs) [10,12]. Morphological analysis of SKPs by use of phase-contrast microscopy revealed that after the treatment of SKPs with PTD-VHL peptide these cells showed significant neurite outgrowth by 24 hours after the start of treatment, whereas a peptide from another part of the sequence of the VHL protein or the PTD-alone peptide did not elicit such outgrowth. Immunocytochemical analysis of NFM in SKPs 7 days after treatment with PTD-VHL-peptide indicated significantly higher expression of these neurofilaments in PTD-VHL peptide-treated cells than in the control ones [10] (Figure 1). Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed up-regulation of basic helix-loop-helix (bHLH) determination factors (neurogenin3; Ngn3) and that of bHLH differentiation factor (NeuroD) as well as down-regulation of inhibitory bHLH (Hes1) in PTD-VHL peptide treated SKPs compared with their respective levels in the control cells [10].

In a study on human hair follicle stem cells (FSCs), neuronal differentiation of the stem cells was examined after the intracellular delivery of PTD-VHL peptide [13]. Two days after delivery of 1-µM peptide, observation by phase-contrast microscopy showed that most cells extended neurite-like cellular processes; whereas the control cells receiving just the PTD peptide elaborated significantly fewer of these processes. In this same immunocytochemical in vitro study, the PTD-VHL peptide-treated FSCs highly expressed various neuronal markers (MAP2, NFM, NFH, and Tuj-1); whereas those treated with PTD peptide alone showed only low expression of these markers [13].

In addition, neuronal differentiation of rat bone marrow mesenchymal stem cells (MSCs) by intracellular delivery of PTD-VHL peptide was examined in vitro [11]. One week after the intracellular delivery of the peptide, MSCs changed their morphology into multiple process-bearing neuron-like cells and expressed neuronal lineage markers, Tuj-1 and MAP-2. PTD-VHL peptide-bearing MSCs showed significantly higher rates of expression of both Tuj-1 and NFH than the control peptide-treated ones. Quantitative RT-PCR evaluation indicated that the MSCs with transferred PTD-VHL peptide expressed significantly more NFH mRNA or MAP2 mRNA than the ones treated with the control peptide. Western blot analysis indicated that the PTD-VHL peptide-bearing MSCs distinctly expressed NFH and MAP2 proteins, whereas the cells treated with the PTD peptide alone scarcely did so [11].

Transplantation of somatic stem cells transfected with VHL gene and that of PTD-VHL peptide-treated ones into the rodent brain

Yamada et al. transplanted somatic stem cells into the rodent brain to examine whether these cells could differentiate into functional neuronal cells and repair the damage caused by neuronal diseases [7]. First of all, after NPCs had been transfected with VHL protein-expressing adenovirus vectors, the cells were transplanted into the striatum of rats modeling Parkinson’s disease (PD). Prior to the transplantation, these cells were labeled with bromodeoxyuridine (BrdU). The results revealed that numerous BrdU-tyrosine hydroxylase (TH) double-labeled cells could be seen close to the transplant site, showing that the transplanted cells had efficiently generated new dopaminergic neurons within the host striatum. Moreover, all of the animals transfected with NPCs with the VHL gene showed a remarkable decrease in apomorphine-induced rotations. These findings thus indicate that NPCs transfected with the VHL gene could efficiently differentiate into dopaminergic neurons [7].

In other studies exploring the possibility that SKPs could survive and function in vivo, SKPs treated with PTD-VHL peptide were transplanted into the striatum of PD model rats [10,12]. The transplanted SKPs differentiated into not only TH- or dopamine transporter (DAT)-positive cells in the rat brain, but also secreted dopamine and repaired symptoms (e.g., apomorphine-induced rotations) in this PD model (unilateral 6-OHDA-lesioned rats) [12]. Two weeks after the 6-OHDA lesioning, the rats were depleted of dopaminergic innervations in their ipsilateral striatum and exhibit a characteristic rotation behavior as a response to the apomorphine challenge [12]. Functional analysis of this apomorphine-induced rotation behavior revealed that rats in the PTD-VHL-peptide-treated SKP group showed a slight but significantly greater improvement at 4 weeks after the transplantation than the rats in the non-treated SKP group or sham-operation group. To evaluate the survival and morphological maturation of SKPs grafted in the striatum of PD model rats, the cells were prelabeled with red fluorescence before grafting. The animals were then sacrified for histological analysis 8 weeks after the engraftment. No tumor formation by the grafts was found in these animals [10]. Immunohistochemical analysis of sections by use of confocal microscopy revealed that more than 5% of the fluorescence-labeled transplanted cells survived in the PD rats. In the PTD-VHL-peptide-SKP group, fluorescence -labeled cells showed high positive rate for NeuN as well as for TH; and the sprouting of dopaminergic neurons surrounding the fluorescence-labeled cells was enhanced; whereas in the sections prepared from the naive SKP group, the fluorescence labeled cells showed a low positive rate for NeuN as well as for TH [10] (Figure 1).

PTD-VHL peptide-bearing FSCs and control ones were prelabelled with red fluorescence and separately implanted into rodent brains [13]. Three weeks later, after perfusion/ fixation, the brains were frozen with liquid nitrogen and sectioned. The number of surviving implanted cells (red fluorescence -prelabelled cells) among the implanted PTD-VHL-peptide treated FSCs was significantly greater than that of the cells treated with PTD alone. In addition, prelabelled cells expressing Tuj-1 represented 38.8% ± 3.5% of the PTD-VHL peptide-treated cell population, which percentage was significantly greater (p < 0.01) than the 9.3% ± 1.5% found for the one treated with PTD peptide alone [13].

Transplantation of somatic stem cells into the spinal cord

Immunohisto chemical results obtained by confocal analysis four weeks after transplantation of NSCs into striatum revealed that the number of surviving (red fluorescence-prelabeled cells) PTD-VHL-peptide-treated NSCs was significantly greater than that of the non-treated cells (P<0.05). In addition, the PTD-VHL-peptide-treated fluorescence-prelabeled NSCs co-localized more with neuronal markers than with a glial marker suggesting that these NSCs had differentiated into neurons rather than into astrocytes in the injured spinal cord [9].

Similarly, four weeks after transplantation of MSCs into the rat spinal cord, confocal analysis of MSCs treated with PTDVHL peptide or PTD peptide alone showed that the number of the surviving cells (red fluorescence-prelabeled cells) was significantly greater for the former than for the latters (P<0.01). The data also indicated that more PTD-VHL peptide-MSCs had differentiate into NFH-positive cells than the cells treated with PTD peptide alons [11].

Correlation of functional recovery in PD-model rats with dopamine levels in the lesioned striatum

The behavior of a novel population of VHL gene-transfected NPCs compared with that of non-transfected NPCs was investigated after their implantation into the adult rat striatum previously lesioned by 6-OHDA [7]. Most non-transfected transplanted NPCs differentiated into astrocytes, and only a few of them differentiated into neurons. In contrast, VHL genetransfected-NPCs differentiated into neurons, with more TH-positive cells formed in vivo than in vitro. The physiological function of TH-positive cells generated from these latter NPCs was assessed in this study by analysis of recovery from apomorphine induced rotation behavior in a rat model of PD. All of the animals transplanted with VHL gene-transfected-NPCs showed a remarkable decrease in apomorphine-induced rotations for up to 16 weeks. In contrast, no behavioral improvements were noted in rats after sham surgery or after transplantation with non-transfected NPCs. Therefore, the behavioral improvement after transplantation of the former NPCs was considered to relate primarily to the survival of grafted TH-positive cells, neuritic outgrowth with synaptic connectivity, and graft-derived dopamine production. As the data indicated that numerous TH-positive cells hadodifferentiated from the VHL gene-transfected NPCs, this method using NPCs transfected with the VHL gene can be expected to likely supply a sufficient number of dopaminergic neurons to treat PD patients [7].

In other studies, apomorphine-induced rotational behavior of 6-OHDA-lesioned rats in the group implanted with PTD-VHL peptide-treated SKPs (PTD-VHL-SKP group) was compared with that in control group rats [10,12]. In the PTD-VHL-SKP group, the mean number of rotations per minute was significantly decreased. Extracellular dopamine levels in the lesioned striatum post-transplantation were evaluated by measuring the dopamine content in dialysates. Dopamine levels in the PTD-VHL-SKP group were significantly greater than those in the non-treated SKP group, thus suggesting that transplantation of PTD-VHL peptide-treated SKPs increased the dopamine concentration in the lesioned striatum [12]. The reduction in the numbersof apomorphine-induced rotations in each rat was plotted against the dopamine level in the engrafted striatum, and linear-regression analysis was performed. The analysis indicated that these 2 parameters were significantly positively correlated [12]. Therefore, these data suggest that the increased dopamine production induced by transplantation of SKPs treated with PTD-VHL peptide contributed to the behavioral recovery in the PD rats [10,12].

Evaluation with Basso-Beattie-Bresnhan (BBB) locomotion scores in rats transplanted with NSCs ord MSCs

In rats transplanted with NSCs, at 1 week after spinal cord injury,the BBB scores, which reflect rat locomotor function, were 0 points in the sham-operated rats;,and at 1 week after transplantation, there were no significant differences in the BBB scores of the NSCstransplantation groups. From 35 days after transplantation, in the BBB scores, a significant difference was recognized between rats transplanted with PTD-VHL-peptide-treated NSCs and rats transplanted with non-treated NSCs (P<0.05) or sham-operated rats (P<0.01) [9].

Similarly, in rats transplanted with MSCs, postoperative locomotor function evaluated in terms of the BBB score was judged as 0 points in each group 7 days after surgery. The rats transplanted with the PTD-VHL peptide-treated MSCs showed significant improvement of their BBB scores (P<0.01) compared with the score for each of the other groups [11].


Expression of VHL protein was progressively observed in cultured NPCs. VHL protein was initially undetectable in freshly harvested E12 cells, which were mostly nestin positive. By day 14, expression of VHL protein was increasingly evident. In addition, expression of VHL protein was correlated with expression of MAP-2, and VHL protein and MAPs were expressed in the same cells. However, expression of VHL protein was shown in the nucleus and the cytoplasm, whereas MAPs expression was shown in the cytoplasm and the dendrites. Their expression in the same cell type suggested that the VHL protein might be involved in central nervous system (CNS) development [5]. The study on VHL gene transfection of NPCs revealed that the VHL protein potently induced neuronal differentiation. In contrast, suppression of the VHL gene inhibited neuronal differentiation and promoted cell-cycle transition in NPCs [5]. In addition, it has been shown that expression of the VHL protein correlates with neuronal differentiation but not with glial differentiation [5]. It was also shown that VHL gene transfection can efficiently produce not only MAP-2-positive cells but also TH-positive cells [7]. Moreover, when GDNF was added to the medium, dopamine production remarkably increased. This fact shows that VHL and GDNF acted synergistically to direct NPC differentiation into dopaminergic neurons [7]. A previous report showed that progenitor cells isolated from the developing human brain, and transplanted into the rat striatum, produced very few TH-positive cells, and those cells in the striatum mostly differentiated into astrocytes [16].

All of the animals transplanted with VHL gene-transfected NPCs showed a remarkable behavioral improvement. In contrast, no behavioral improvements were noted in rats after sham surgery or after transplantation with non-treated NPCs. Therefore, the behavioral improvement after transplantation of VHL gene- transfected NPCs is suggested to be related to the survival of grafted TH-positive cells, neuritic outgrowth with synaptic connectivity, and graft- derived dopamine production. The finding that numerous TH-positive cells differentiated from the grafted VHL gene-transfected NPCs suggestd that there would likely be an adequate supply of dopaminergic neurons formed for regenerative therapy ot PD patients.

To improve functional recovery after cell transplantation into the injured spinal cord, it is critical that transplanted cells survive and differentiate into neuronal cell lineages beneficial for remyelination, functional synaptic replacement, neurotrophic factor delivery, and axon elongation, all of which contribute to the promotion of spinal cord regeneration [2]. Therefore a strategy using implantation of neuronal lineage-restricted stem cells has been advocated, and it was shown that neuronal differentiation on NSCs by intracellular delivery of PTD-VHL peptide into NSCs could be used as an alternative method to the gene transfection [11]. VHL-peptide-treated NSCs show s higher rate of survival when engrafted into the spinal cord than non-treated NSCs. This higher survival rate of transplanted VHL-peptide delivered NSCs was also recognized in the intracerebral transplantation of VHL gene-transfectedd NSCs [7]. These neurally differentiated NSCs might become acclimatized to the environment of the neuronal tissue. In addition, statistically significant behavioral difference between rats transplanted with PTD-VHL-peptide-treated NSCs and rats with non-treated NSCs was found in terms of locomotion. This fact suggested that these cells brought about an improvement in the motor function in rats with acute spinal cord injury. The finding of the neuronal differentiation of NSCs through VHL-peptide transfer could well contribute to the cure of spinal cord injuries. Similarly, neuronal differentiation of MSCs before cell transplantation is fundamental for therapy aimed at regeneration. Compared with NPCs/NSCs [8,9] SKPs [10] and MSCs showed less neuronal differentiation by intracellular delivery of the VHL peptide Untreated naive MSCs were unsatisfactorily differentiated into neuron-like cells when transplanted into the spinal cord and were insufficient in causing behavorial recovery of model rats with spinal cord injury, where as VHL peptide-treated MSCs were significantly more differentiated into neuron-like cells and sufficiently recovered normal behavior. These results suggest that neuronal differentiation of MSCs before grafting for spinal cord injury could contribute to repair of the injured spinal cord. In comparison with other reports on the transplantation of treated MSCs for the repair of spinal cord injuries, the method using VHL peptide showed equal or greater functional recovery in terms of the BBB locomotor scale than other methods [11].

SKPs are the ideal precursor cell populations that can be derived in an autologous fashion from small amounts of accessible tissue biopsies and are pluripotent somatic stem cells capable of differentiating into both neural and mesodermal progenies. Ine our previous studies [10,12], SKPs with specific induction via PTD-VHL peptide were shown to differentiate into dopamine neuron-like cells in vitro and after being transplanted into PD model rats. Suchsstudiesdcontribute to cell therapy for PD [17,18]..

In addition, neuronal differentiation of human FSCs occurred when the PTD-VHL peptide was intracellularly delivered into these [13]. These cells are promising as donor cells for the treatment of intractable neuronal diseases. However, if these cells without neuronal differentiation are implanted, they scarcely survive or differentiate into functional neuronal cells, similar to the case of other stem cells. Therefore, before implantation for cell therapy of intractable neuronal diseases, such cells would be required to differentiate into neuronal cells. Intractable neuronal diseases such as PD disease develop more frequently in elderly patients. Therefore, it is significant that multipotent somatic stem cells obtained from elderly humans can be used as donor cells for cell transplantation therapy [13].

The neuronal-differentiation method using PTD-VHL peptide is very simple because only the PTD-VHL peptide needs to be added to basic medium lacking serum, and it is also rapid compared with previously reported methods using various neurotrophic factors and other neuronal- differentiation-inducing agents. Thus, the use of PTD-VHL peptide is recommended for the neuronal differentiation of multipotent somatic stem cells.

Although there is much evidence that intracellularly delivered VHL peptide induced neuronal differentiation of various somatic stem cells, the molecular mechanism underlying this induction has not been clarified. The VHL peptide was designed to specifically bind to elongin C. Activation of elongin C by VHL peptide is necessary and sufficient for formation of the elongin BC complex and subsequent ubiquitin-dependent signal regulation for cellular growth and differentiation [12]. In the crystal structure, the VHL peptide forms a helix that fits into a concave surface present on elongin C. It seems likely that inhibition of elongin A contributes to function of VHL protein, and that competition between VHL peptide and elongin A may be related to the effects of the peptide on neuronal differentiation of somatic stem cells. In addition, the elongin BC complex binds to various kinds of proteins that can induce the ubiquitination and degradation of Stat 3 [12]. It has been reported that Stat3 promotes astrocytic differentiation, and that degradation of Stat 3 directly inhibits astrocytic differentiation and accelerates neuronal differentiation [19]. Further study will be required to investigate whether those pathways involving Stat 3 play an important role in neuronal differentiation induced by the VHL peptide [12].

In conclusion, it was shown thate when somatic stem cells treated in vitro with the VHL gene or PTD-VHL peptide were implanted into the rat brain or spinal cord, they differentiated into neuronal marker-positive cells. The neuronal differentiation method using PTD-VHL peptide is very simple, rapid, and superior to the methods using transfection with the VHL gene or neuronal differentiation-inducing agents. Thus, the use of PTD-VHL peptide for the neuronal differentiation of multipotent somatic stem cells is to be recommended.


1. Lonser RR, Glenn GM, Walther M, Chew EY, Libutti SK, Linehan WM, et al. von Hippel-Lindau disease. Lancet. 2003; 361: 2059-2067.

2. Latif F, Tory K, Gnarra J, Yao M, Duh FM, Orcutt ML, et al. Identification of the von Hippel-Lindau disease tumor suppressor gene. Science. 1993; 260: 1317-1320.

3. Duan DR, Pause A, Burgess WH, Aso T, Chen DY, Garrett KP, et al. Inhibition of transcription elongation by the VHL tumor suppressor protein. Science. 1995; 269: 1402-1406.

4. Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 1999; 399: 271-275.

5. Kanno H, Saljooque F, Yamamoto I, Hattori S, Yao M, Shuin T, et al. Role of the von Hippel-Lindau tumor suppressor protein during neuronal differentiation. Cancer Res. 2000; 60: 2820-2824.

6. Los M, Jansen GH, Kaelin WG, Lips CJ, Blijham GH, Voest EE. et al. Expression pattern of the von Hippel-Lindau protein in human tissues. Lab Invest. 1996; 75: 231-238.

7. Yamada H, Dezawa M, Shimazu S, Baba M, Sawada H, Kuroiwa Y, et al. Transfer of the von Hippel-Lindau gene to neuronal progenitor cells in treatment for Parkinson’s disease. Ann Neurol. 2003; 54: 352-359.

8. Kanno H, Nakano S, Kubo A, Mimura T, Tajima N, Sugimoto N. Neuronal differentiation of neural progenitor cells by intracellular delivery of synthetic oligopeptide derived from Von Hippel-Lindau protein. Protein Pept Lett. 2009; 16: 1291-1296.

9. Maeda K, Kanno H, Yamazaki Y, Kubo A, Sato F, Yamaguchi Y, et al. Transplantation of Von Hippel-Lindau peptide delivered neural stem cells promotes recovery in the injured rat spinal cord. Neuroreport. 2009; 20: 1559-1563.

10. Kubo A, Yoshida T, Kobayashi N, Yokoyama T, Mimura T, Nishiguchi T, et al. Efficient generation of dopamine neuron-like cells from skin-derived precursors with a synthetic peptide derived from von Hippel-Lindau protein. Stem Cells Dev. 2009; 18: 1523-1532.

11. Yamazaki Y, Kanno H, Maeda K, Yoshida T, Kobayashi N, Kubo A, et al. Engrafted VHL peptide-delivered bone marrow stromal cells promote spinal cord repair in rats. Neuroreport. 2010; 21: 287-292.

12. Higashida T, Jitsuki S, Kubo A, Mitsushima D, Kamiya Y, Kanno H. Skin-derived precursors differentiating into dopaminergic neuronal cells in the brains of Parkinson disease model rats. J Neurosurg. 2010; 113: 648-655.

13. Kanno H, Kubo A, Yoshizumi T, Mikami T, Maegawa J. Isolation of Multipotent nestin-expressing stem cells derived from the epidermis of elderly humans and TAT-VHL peptide-mediated neuronal differentiation of these cells. Int J Mol Sci. 2013; 14: 9604-9617.

14. Takahashi J, Palmer TD, Gage FH. Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult-derived neural stem cell cultures. J Neurobiol. 1999; 38: 65-81.

15. Kanno H, Sato H, Yokoyama TA, Yoshizumi T, Yamada S. The VHL tumor suppressor protein regulates tumorigenicity of U87-derived glioma stem-like cells by inhibiting the JAK/STAT signaling pathway. Int J Oncol. 2013; 42: 881-886.

16. Svendsen CN, Caldwell MA, Shen J, ter Borg MG, Rosser AE, Tyers P, et al. Long-term survival of human central nervous system progenitor cells transplanted into a rat model of Parkinson’s disease. Exp Neurol. 1997; 148: 135-146.

17. Nishino H, Hida H, Takei N, Kumazaki M, Nakajima K, Baba H. Mesencephalic neural stem (progenitor) cells develop to dopaminergic neurons more strongly in dopamine-depleted striatum than in intact striatum. Exp Neurol. 2000; 164: 209-214.

18. Ho A, Schwarze SR, Mermelstein SJ, Waksman G, Dowdy SF. Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer Res. 2001; 61: 474-477.

19. Takizawa T, Yanagisawa M, Ochiai W, Yasukawa K, Ishiguro T, Nakashima K, et al. Directly linked soluble IL-6 receptor-IL-6 fusion protein induces astrocyte differentiation from neuroepithelial cells via activation of STAT3. Cytokine. 2001; 13: 272-279.

Received : 29 Oct 2013
Accepted : 21 Nov 2013
Published : 23 Nov 2013
Annals of Otolaryngology and Rhinology
ISSN : 2379-948X
Launched : 2014
JSM Schizophrenia
Launched : 2016
Journal of Nausea
Launched : 2020
JSM Internal Medicine
Launched : 2016
JSM Hepatitis
Launched : 2016
JSM Oro Facial Surgeries
ISSN : 2578-3211
Launched : 2016
Journal of Human Nutrition and Food Science
ISSN : 2333-6706
Launched : 2013
JSM Regenerative Medicine and Bioengineering
ISSN : 2379-0490
Launched : 2013
JSM Spine
ISSN : 2578-3181
Launched : 2016
Archives of Palliative Care
ISSN : 2573-1165
Launched : 2016
JSM Nutritional Disorders
ISSN : 2578-3203
Launched : 2017
Annals of Neurodegenerative Disorders
ISSN : 2476-2032
Launched : 2016
Journal of Fever
ISSN : 2641-7782
Launched : 2017
JSM Bone Marrow Research
ISSN : 2578-3351
Launched : 2016
JSM Mathematics and Statistics
ISSN : 2578-3173
Launched : 2014
Journal of Autoimmunity and Research
ISSN : 2573-1173
Launched : 2014
JSM Arthritis
ISSN : 2475-9155
Launched : 2016
JSM Head and Neck Cancer-Cases and Reviews
ISSN : 2573-1610
Launched : 2016
JSM General Surgery Cases and Images
ISSN : 2573-1564
Launched : 2016
JSM Anatomy and Physiology
ISSN : 2573-1262
Launched : 2016
JSM Dental Surgery
ISSN : 2573-1548
Launched : 2016
Annals of Emergency Surgery
ISSN : 2573-1017
Launched : 2016
Annals of Mens Health and Wellness
ISSN : 2641-7707
Launched : 2017
Journal of Preventive Medicine and Health Care
ISSN : 2576-0084
Launched : 2018
Journal of Chronic Diseases and Management
ISSN : 2573-1300
Launched : 2016
Annals of Vaccines and Immunization
ISSN : 2378-9379
Launched : 2014
JSM Heart Surgery Cases and Images
ISSN : 2578-3157
Launched : 2016
Annals of Reproductive Medicine and Treatment
ISSN : 2573-1092
Launched : 2016
JSM Brain Science
ISSN : 2573-1289
Launched : 2016
JSM Biomarkers
ISSN : 2578-3815
Launched : 2014
JSM Biology
ISSN : 2475-9392
Launched : 2016
Archives of Stem Cell and Research
ISSN : 2578-3580
Launched : 2014
Annals of Clinical and Medical Microbiology
ISSN : 2578-3629
Launched : 2014
JSM Pediatric Surgery
ISSN : 2578-3149
Launched : 2017
Journal of Memory Disorder and Rehabilitation
ISSN : 2578-319X
Launched : 2016
JSM Tropical Medicine and Research
ISSN : 2578-3165
Launched : 2016
JSM Head and Face Medicine
ISSN : 2578-3793
Launched : 2016
JSM Cardiothoracic Surgery
ISSN : 2573-1297
Launched : 2016
JSM Bone and Joint Diseases
ISSN : 2578-3351
Launched : 2017
JSM Bioavailability and Bioequivalence
ISSN : 2641-7812
Launched : 2017
JSM Atherosclerosis
ISSN : 2573-1270
Launched : 2016
Journal of Genitourinary Disorders
ISSN : 2641-7790
Launched : 2017
Journal of Fractures and Sprains
ISSN : 2578-3831
Launched : 2016
Journal of Autism and Epilepsy
ISSN : 2641-7774
Launched : 2016
Annals of Marine Biology and Research
ISSN : 2573-105X
Launched : 2014
JSM Health Education & Primary Health Care
ISSN : 2578-3777
Launched : 2016
JSM Communication Disorders
ISSN : 2578-3807
Launched : 2016
Annals of Musculoskeletal Disorders
ISSN : 2578-3599
Launched : 2016
Annals of Virology and Research
ISSN : 2573-1122
Launched : 2014
JSM Renal Medicine
ISSN : 2573-1637
Launched : 2016
Journal of Muscle Health
ISSN : 2578-3823
Launched : 2016
JSM Genetics and Genomics
ISSN : 2334-1823
Launched : 2013
JSM Anxiety and Depression
ISSN : 2475-9139
Launched : 2016
Clinical Journal of Heart Diseases
ISSN : 2641-7766
Launched : 2016
Annals of Medicinal Chemistry and Research
ISSN : 2378-9336
Launched : 2014
JSM Pain and Management
ISSN : 2578-3378
Launched : 2016
JSM Women's Health
ISSN : 2578-3696
Launched : 2016
Clinical Research in HIV or AIDS
ISSN : 2374-0094
Launched : 2013
Journal of Endocrinology, Diabetes and Obesity
ISSN : 2333-6692
Launched : 2013
Journal of Substance Abuse and Alcoholism
ISSN : 2373-9363
Launched : 2013
JSM Neurosurgery and Spine
ISSN : 2373-9479
Launched : 2013
Journal of Liver and Clinical Research
ISSN : 2379-0830
Launched : 2014
Journal of Drug Design and Research
ISSN : 2379-089X
Launched : 2014
JSM Clinical Oncology and Research
ISSN : 2373-938X
Launched : 2013
JSM Bioinformatics, Genomics and Proteomics
ISSN : 2576-1102
Launched : 2014
JSM Chemistry
ISSN : 2334-1831
Launched : 2013
Journal of Trauma and Care
ISSN : 2573-1246
Launched : 2014
JSM Surgical Oncology and Research
ISSN : 2578-3688
Launched : 2016
Annals of Food Processing and Preservation
ISSN : 2573-1033
Launched : 2016
Journal of Radiology and Radiation Therapy
ISSN : 2333-7095
Launched : 2013
JSM Physical Medicine and Rehabilitation
ISSN : 2578-3572
Launched : 2016
Annals of Clinical Pathology
ISSN : 2373-9282
Launched : 2013
Annals of Cardiovascular Diseases
ISSN : 2641-7731
Launched : 2016
Journal of Behavior
ISSN : 2576-0076
Launched : 2016
Annals of Clinical and Experimental Metabolism
ISSN : 2572-2492
Launched : 2016
Clinical Research in Infectious Diseases
ISSN : 2379-0636
Launched : 2013
JSM Microbiology
ISSN : 2333-6455
Launched : 2013
Journal of Urology and Research
ISSN : 2379-951X
Launched : 2014
Journal of Family Medicine and Community Health
ISSN : 2379-0547
Launched : 2013
Annals of Pregnancy and Care
ISSN : 2578-336X
Launched : 2017
JSM Cell and Developmental Biology
ISSN : 2379-061X
Launched : 2013
Annals of Aquaculture and Research
ISSN : 2379-0881
Launched : 2014
Clinical Research in Pulmonology
ISSN : 2333-6625
Launched : 2013
Journal of Immunology and Clinical Research
ISSN : 2333-6714
Launched : 2013
Annals of Forensic Research and Analysis
ISSN : 2378-9476
Launched : 2014
JSM Biochemistry and Molecular Biology
ISSN : 2333-7109
Launched : 2013
Annals of Breast Cancer Research
ISSN : 2641-7685
Launched : 2016
Annals of Gerontology and Geriatric Research
ISSN : 2378-9409
Launched : 2014
Journal of Sleep Medicine and Disorders
ISSN : 2379-0822
Launched : 2014
JSM Burns and Trauma
ISSN : 2475-9406
Launched : 2016
Chemical Engineering and Process Techniques
ISSN : 2333-6633
Launched : 2013
Annals of Clinical Cytology and Pathology
ISSN : 2475-9430
Launched : 2014
JSM Allergy and Asthma
ISSN : 2573-1254
Launched : 2016
Journal of Neurological Disorders and Stroke
ISSN : 2334-2307
Launched : 2013
Annals of Sports Medicine and Research
ISSN : 2379-0571
Launched : 2014
JSM Sexual Medicine
ISSN : 2578-3718
Launched : 2016
Annals of Vascular Medicine and Research
ISSN : 2378-9344
Launched : 2014
JSM Biotechnology and Biomedical Engineering
ISSN : 2333-7117
Launched : 2013
Journal of Hematology and Transfusion
ISSN : 2333-6684
Launched : 2013
JSM Environmental Science and Ecology
ISSN : 2333-7141
Launched : 2013
Journal of Cardiology and Clinical Research
ISSN : 2333-6676
Launched : 2013
JSM Nanotechnology and Nanomedicine
ISSN : 2334-1815
Launched : 2013
Journal of Ear, Nose and Throat Disorders
ISSN : 2475-9473
Launched : 2016
JSM Ophthalmology
ISSN : 2333-6447
Launched : 2013
Journal of Pharmacology and Clinical Toxicology
ISSN : 2333-7079
Launched : 2013
Annals of Psychiatry and Mental Health
ISSN : 2374-0124
Launched : 2013
Medical Journal of Obstetrics and Gynecology
ISSN : 2333-6439
Launched : 2013
Annals of Pediatrics and Child Health
ISSN : 2373-9312
Launched : 2013
JSM Clinical Pharmaceutics
ISSN : 2379-9498
Launched : 2014
JSM Foot and Ankle
ISSN : 2475-9112
Launched : 2016
JSM Alzheimer's Disease and Related Dementia
ISSN : 2378-9565
Launched : 2014
Journal of Addiction Medicine and Therapy
ISSN : 2333-665X
Launched : 2013
Journal of Veterinary Medicine and Research
ISSN : 2378-931X
Launched : 2013
Annals of Public Health and Research
ISSN : 2378-9328
Launched : 2014
Annals of Orthopedics and Rheumatology
ISSN : 2373-9290
Launched : 2013
Journal of Clinical Nephrology and Research
ISSN : 2379-0652
Launched : 2014
Annals of Community Medicine and Practice
ISSN : 2475-9465
Launched : 2014
Annals of Biometrics and Biostatistics
ISSN : 2374-0116
Launched : 2013
JSM Clinical Case Reports
ISSN : 2373-9819
Launched : 2013
Journal of Cancer Biology and Research
ISSN : 2373-9436
Launched : 2013
Journal of Surgery and Transplantation Science
ISSN : 2379-0911
Launched : 2013
Journal of Dermatology and Clinical Research
ISSN : 2373-9371
Launched : 2013
JSM Gastroenterology and Hepatology
ISSN : 2373-9487
Launched : 2013
Annals of Nursing and Practice
ISSN : 2379-9501
Launched : 2014
JSM Dentistry
ISSN : 2333-7133
Launched : 2013
Author Information X