Conversion of Adipose Tissue-Derived Mesenchymal Stem Cells to Neural Stem Cell-Like Cells by a Single Transcription Factor,Sox2

Introduction

Adipose tissue provides attractive, easily accessible adult candidate cells for regenerative medicine and can be isolated from both males and females at different ages, because obesity is a common problem and liposuction is a relatively safe and popular procedure. Adipose tissue–derived mesenchymal stem cells (ADSCs) have multipotency, can undergo self-renewing divisions, and possess the capacity to differentiate into osteogenic, chondrogenic, and adipogenic cell lineages (Qin et al., 2014; Zeve et al., 2009; Zuk et al., 2001). In addition, human and mouse adipose tissue–derived stem cells not only can be reprogrammed to induced pluripotent stem cells (iPSCs) with substantially higher efficiencies than those reported for human and mouse fibroblasts (Sugii et al., 2010), but they also have stronger proliferation and differentiation capabilities in vitro than skin fibroblasts (Rodeheffer et al., 2008; Zuk et al., 2001). In addition, we have recently reported that cloned mice and embryonic stem cells (ESCs) can be produced from adipose tissue–derived cells (Qin et al., 2013, 2015) and revealed that these cells possess good genetic stability. However, as mesodermal multipotent stem cells, whether the ADSCs can be directly converted into neural stem cells (NSCs) so far has not been demonstrated.

By transcription factor transduction, somatic cells can not only be reprogrammed to iPSCs (Takahashi and Yamanaka, 2006), but also directly converted from one cell type to another, such as conversion of fibroblasts into NSCs (Han et al., 2012) or neurons (Vierbuchen et al., 2010). Recently, Ring et al. reported the generation of induced neural stem cells (iNSCs) from mouse and human fibroblasts by direct reprogramming with a single transcription factor, Sox2 (Ring et al., 2012). NSCs have self-renewal capacity, can continue to be cultured and expanded in serum-free medium in vitro, and can differentiate into essentially all kinds of neurons. Unlike ESCs and iPSCs, iNSCs are not tumorigenic (Han et al., 2012; Ring et al., 2012). In view of the above advantages of ADSCs and NSCs, we wanted to convert ADSCs to induced NSC-like (iNSC-like) cells with a single factor, Sox2.

In this study, we used our previously purified and characterized ADSCs that expressed mesenchymal stem cell (MSC)-specific markers and possessed osteogenic, chondrogenic, and adipogenic differentiation potential (Qin et al., 2014). We found that the ADSCs maintained in monolayer or in spheres did not express Sox2 and Pax6 genes and proteins and did not differentiate to neuron-like cells. However, after they were infected with the retrovirus expressing Sox2, they showed classic NSC morphology and neural spheres and expressed NSC markers, including Sox2 and Pax6, on the basis of RT-PCR and immunostaining analyses. In addition, the NSC-like cells displayed the ability to differentiate into neuron-like cells, expressing neuronal markers such as Tuj1 and MAP2 genes and proteins. The present study indicates that ADSCs can be converted into NSC-like cells with a single transcription factor, Sox2.

Materials and Methods

Results

Generation and characterization of iNSC-like cells from ADSCs

In this study, we used our previously purified and characterized ADSC cell line, which expressed specific MSC markers and possessed osteogenic, chondrogenic, and adipogenic differentiation potential (Qin et al., 2014). When the ADSCs were cultured in normal medium (DMEM with 10% FBS), they exhibited a typical fibroblast-like morphology (Fig. 1A, left). However, when ADSCs were trypsinized and replated in NSC medium (see Materials and Methods) and maintained under this culture condition for 6–7 days, they formed spheres, called ADSC spheres (Fig. 1A, middle).

OPEN IN VIEWER

FIG. 1.

Generation and characterization of iNSC-like cells from ADSCs. (A) ADSCs cultured in normal medium exhibited a typical fibroblast-like morphology (left). However, ADSCs cultured in NSC medium could form spheres, called ADSC spheres (middle). ADSCs infected with Sox2 retrovirus cultured in NSC medium exhibited a typical NSC-like morphology, showed neural spheres, and could be passaged continuously (right). These cells were called iNSC-like cells. (B) RT-PCR analysis showed that ADSCs, ADSC spheres, and iNSC-like cells all expressed the Nestin gene, but only iNSC-like cells expressed the Sox2 and Pax6 genes. Scale bar, 50 μm.

Given that Sox2 is reported to highly expressed in NSCs (Sarkar and Hochedlinger, 2013) and can convert somatic cells into NSCs (Ring et al., 2012), we hypothesize that transduction of ADSCs might induce them to become iNSCs. We infected ADSCs with 1 mL of Sox2 retrovirus for 24 h and then cultured the cells in NSC medium. After culture for 6–7 days, these infected cells were digested and dissociated using Accutase and replated in NSC medium. After another 6–7 days in culture, these cells exhibited a typical NSC-like morphology (Fig. 1A, right), formed neural spheres, and could be passaged continuously. These cells were called iNSC-like cells.

To verify whether these cells had the characteristics of NSCs, we first performed RT-PCR analysis to measure expression levels of NSC-related genes, including Nestin, Sox2, and Pax6. We found that while the Nestin gene was expressed by some of the ADSCs, ADSC-sphere cells, and iNSC-like cells, Sox2 and Pax6 genes were only expressed by iNSC-like cells (Fig. 1B).

Next, we used immunofluorescent staining to determine expression levels of NSC-related proteins. Our results revealed that ADSCs, ADSC-spheres, and iNSC-like cells all expressed Nestin, but only iNSC-like cells expressed Sox2 and Pax6 proteins (Fig. 2). In sharp contrast, the naïve ADSCs and ADSC spheres did not express Sox2 and Pax6 genes and proteins (Figs. 1B and 2).

OPEN IN VIEWER

FIG. 2.

Immunofluorescence analysis of iNSC-like cells. Immunostaining showed that ADSCs, ADSC spheres, and iNSC-like cells all expressed Nestin proteins, but only iNSC-like cells expressed Sox2 and Pax6 proteins. In sharp contrast, ADSCs and ADSC spheres did not express these proteins. Scale bar, 50 μm.

Neural differentiation of iNSC-like cells in vitro

To verify whether the iNSC-like cells could differentiate into neural cells, we seeded these cells on to polyornithine/laminin-coated plates in neural differentiation medium (see Materials and Methods). After culture for 2–4 weeks, iNSCs-like cells differentiated into typical neuron-like cells in shape (Fig. 3A, bottom). By RT-PCR analysis, we found that these differentiated neuron-like cells indeed expressed the neuron-related genes Tuj1 and MAP2 (Fig. 3B). Next, we used immunofluorescent staining to study if the differentiated neuron-like cells from iNSCs-like cells also expressed the neuron-related proteins Tuj1 and MAP2. As shown in Figure 4 (bottom panel), they indeed expressed Tuj1 and MAP2. In contrast, ADSC spheres cultured in neural differentiation medium neither displayed neural morphology nor expressed Tuj1 and MAP2 genes and proteins (Fig. 3A, top, Fig, 3B, and Fig. 4, top panel).

OPEN IN VIEWER

FIG. 3.

Neural differentiation of iNSC-like cells in vitro. (A) After culture in neural differentiation medium for 2–4 weeks, iNSC-like cells differentiated into typical neuron-like cells in morphology (bottom). ADSC spheres cultured in neural differentiation medium, however, did not show neuron-like morphology (top). (B) RT-PCR analysis showed that differentiated neuron-like cells from iNSC-like cells expressed the neuron-related genes Tuj1 and MAP2. ADSC spheres maintained in neural differentiation medium did not express these genes. Scale bar, 50 μm.

OPEN IN VIEWER

FIG. 4.

Immunofluorescence analysis of differentiated neuron-like cells from iNSC-like cells. Immunofluorescent staining confirmed that the differentiated neuron-like cells from iNSC-like cells expressed the neuron-related proteins Tuj1 and MAP2 (bottom panel). In contrast, in control cultures, the ADSC spheres did not express these proteins (top panel). Scale bar, 50 μm.

Discussion

To the best of our knowledge, this is the first report to demonstrate the conversion of ADSCs into iNSC-like cells using one transcription factor, Sox2. These ADSCs-derived iNSC-like cells showed classic NSC morphology and neural spheres. RT-PCR and immunostaining analyses revealed that they expressed NSC markers, including Sox2 and Pax6. In addition, the iNSC-like cells displayed the ability to differentiate into neuron-like cells in vitro, expressing the neuronal marker Tuj1 and MAP2 genes and proteins.

Although adipose-derived stem cells can be reprogrammed to iPSCs with substantially higher efficiencies (Sugii et al., 2010), iNSCs were not tumorigenic as compared to ESCs and iPSCs. Furthermore, the iNSCs have self-renewal capacity and can be cultured continuously, expanded in serum-free medium in vitro, and differentiated into all kinds of neurons. Recently, iNSCs can be generated from skin fibroblasts by a direct reprogramming with a single transcription factor, Sox2 (Ring et al., 2012). However, the acquisition of skin cells from patients has certain disadvantages, such as pain and scar formation. As compared to skin cells, ADSCs have advantages, including easy access, because obesity affects health and liposuction is a relatively safe and popular procedure. In addition, ADSCs possess stronger proliferation in vitro than skin fibroblasts (Han et al., 2012; Ring et al., 2012), and our recent study confirmed ADSCs possess good genetic stability (Qin et al., 2013, 2015). So the adipose tissue from patients can provide virtually unlimited donor cells for the conversion.

It is important to note that as mesodermal multipotent stem cells, it is controversial whether the ADSCs could directly differentiate into NSCs (Gurdon and Melton, 2008; Yamanaka and Blau, 2010). Some studies have reported that when ADSCs are cultured in NSC medium, they can form NSC-like neural spheres and even could further differentiate into neural cells (Ahmadi et al., 2012; Anghileri et al., 2008). However, there was a question of whether these cells are really neurons because these investigators did not determine whether these NSC-like neural spheres express the NSC-specific markers Pax6 and Sox2. Importantly, our study showed that although ADSCs and ADSC spheres express Nestin, they do not express Pax6 and Sox2. Normally the mesoderm-derived ADSCs can differentiate into osteoblasts, chondrocysts, and adipocytes, but do not differentiate into the neural ectoderm cells. These results indicated that ADSCs only possess multipotency, not pluripotency. By transduction of the ADSCs with Sox2 gene, these cells become iNSCs, which can then be induced to differentiate into neurons.

At present, the successful methods of reprogramming somatic cells to the totipotent or pluripotent state are either nuclear transfer technique (to form ESCs, Tachibana et al., 2013) or induction of pluripotency by the Yamanaka factors (to form iPSCs; Takahashi and Yamanaka, 2006). In addition, by special transcription factor transduction, somatic cells can be converted from one cell type to another, such as from fibroblasts into NSCs (Han et al., 2012) or neurons (Vierbuchen et al., 2010). Recently, it was shown that by a single transcription factor, Sox2, the fibroblasts could be reprogrammed directly to iNSCs (Ring et al., 2012). This method made the transcription factor-mediated conversion easier. Sox2 is expressed during the earliest stages of ESC differentiation toward the neural lineage in vitro, supporting a role in neural commitment, and it promotes early neuroectodermal fate by directly suppressing key regulators of the alternative mesendodermal fate (Sarkar and Hochedlinger, 2013). The Sox2 functions were considered as a master regulator gene for NSC identity and maintenance.

In this study, we introduced the Sox2 gene into ADSCs by retrovirus, and these cells became iNSCs. Our findings identify another somatic cell source for generation of iNSCs, which are helpful for cell therapy of neurological disorders.