Cell Biology: Research & TherapyISSN: 2324-9293

Research Article, Cell Biol Henderson Nv Vol: 6 Issue: 2

Human Adipose Tissue-Derived Stem Cells Differentiate to Neuronal-like Lineage Cells without Specific Induction

Subathra Radhakrishnan1, Omana Anna Trentz1*, Vinoth Kumar Parthasarathy1 and Shanmugapriya Sellathamby2

1MIOT Institute of Research (MIR), MIOT International, 4/112, Mount poonamalle High Road, Manapakkam, Chennai-89, Tamil Nadu, India

2Department of Biomedical Science, Bharathidasan University, Palkalaiperur, Trichirapalli, Tamil Nadu, India

*Corresponding Author : Omana Anna Trentz,Director
MIOT Institute of Research (MIR), MIOT International, 4/112, Mount poonamalle High Road, Manapakkam, Chennai-89.Tamil Nadu, India
Tel: 044 4200 2288s
E-mail: [email protected]

Received: March 13, 2017 Accepted: May 11, 2017 Published: May 16, 2017

Citation: Radhakrishnan S, Trentz OA, Parthasarathy VK, Sellathamby S (2017) Human Adipose Tissue-Derived Stem Cells Differentiate to Neuronal-like Lineage Cells without Specific Induction. Cell Biol (Henderson, NV) 6:1.doi: 10.4172/2324-9293.1000131

Abstract

Adipose tissue is an attractive source for generating pluripotent stromal cells which can differentiate also into ectodermal cell types. Mesenchymal stem cells are promising candidates for cell therapy and tissue regeneration. Several studies tried to differentiate Adipose tissue-Derived Stem Cells (ADSCs) into neurogenic cells with expression of some neural markers using specific chemical induction protocols. Meanwhile it is proven that toxic chemicals used in the induction media generate artifacts due to cell stress which mimic neural differentiation. In this study the differentiation of human ADSCs into neuronal-like lineage cells without using any induction protocol is investigated. ADSCs were cultured over 65 days and their morphological appearance and the expression of specific neural markers on the posttranscriptional as well as the posttranslational level were studied by RT-PCR and immunocytochemistry. The results suggest the differentiation of ADSCs into characteristic neuronal-like cells with the expression of a wide scale of typical neuronal and glial markers. Whether this is a true reprogramming differentiation and whether this neuronallike lineage cells can produce mature functional neurons must be evaluated by further investigations

Keywords: ADSCs; Differentiation without induction protocol; Neuronal-like lineal cells; Long term cultivation

Introduction

Mesenchymal stem cells (MSCs) are multipotent stromal cells with extensive proliferative potential and the ability to undergo multilineage differentiation like osteoblasts, chondrocytes, adipocytes and myoblasts [1]. Several studies have shown adipose tissue as an alternative source of multipotent stromal cells, which can be obtained by less invasive methods and in larger quantities compared to bone marrow [2-4]. There is also a lot of evidence to suggest that these cells can not only differentiate into mesodermal cells, but also into ectodermal cell types [5].

In 1999 Kopen et al. [6] injected Bone Marrow Stromal Cells (BMSCs) into the lateral ventricle of neonatal mice and found migrating throughout the forebrain and after 12 days developing neural cells in the brain microenvironment. Azizi et al. [7] could also show engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats. Desawa et al. [8] could establish sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated human BMSCs. Zhao et al. [9] grafted human MSCs into the surrounding area of rat cortex infarction and assessed after 6 weeks the transplanted area for sensorimotor function and could also confirm the expression of neuronal markers. Woodbury et al. [10] could expand adult rat and human BMSCs in more than 20 passages and induce with a simple induction protocol neuronal phenotypes with expression of neuronal cell specific markers. Safford et al. [11] could also confirm in murine and human adult Adipose-Derived Stem Cells (ADSCs) the development of neurogenic cells with modified neuronal induction protocols. Sanchez-Ramos et al. [12] demonstrated that adult human and mouse BMSCs can be induced in the presence of Epidermal Growth Factor (EGF) and Brain Derived Neurotrophic Factor (BDNF) into neural cells and express the markers of neuronal precursors protein Nestin, Glial Fibrillary Acidic Protein (GFAP) and Neuronal Specific Nuclear Protein (NeuN). Black and Woodbury [13] revealed 2001 that adult rat and human BMSCs can be induced to differentiate into neuronal phenotypes in subconfluent cells in serum containing medium supplemented with β-mercaptoethanol (β-ME) for 24 hours. However there are also studies suggesting that chemical neuronal induction might be an artifact of cell stress due to toxic chemicals in the induction medium [14,15]. Furthermore some in vitro studies described sporadically that rat and human BMSCs also differentiated spontaneously into cells with neural-like appearance [16,17].

In this study the differentiation of human ADSCs into neuronallike lineage cells is investigated in long-term cultures over 65 days without using any of the established induction protocols containing brain-derived neurotrophic factor (BDNF), other growth factors, chemicals, or combinations of them.

The morphological and phenotypical changes were characterized; the gene- as well as protein-expression of so-called specific neuronal and glial markers detected, and in the discussion compared with published results of trials using induction protocols.

Materials and Methods

Preparation and culture of ADSCs

Cells were harvested out of adipose tissue (white fat of the infrapatellar Hoffa-pad) from 10 patients undergoing knee arthroplasties. All patients had given their informed consent according to the existing local ethical guidelines and were between 57 and 65 years of age (6 females, 4 men).

The fat tissue reached the lab within 30 min in sterile containers with saline solution, and was washed with Dulbecco`s phosphate buffered saline solution (DPBS, Gibco) without calcium and magnesium to remove blood. The specimens were minced and digested in 0.075% of collagen type 1 Pan Biotech 3 to 4 hours, neutralized with standard culture medium [500ml Dulbecco`s modified Eagle`s medium (DMEM) with 10% fetal calf serum (FCS), 60 μg/ml antibiotic-antimycotic (Invitrogen)], and centrifuged for 8min at 1800 rpm. The sediment was resuspended in culture medium and filtered through a 70 μM nylon mesh (BD Falcon, MD, USA) to remove cellular debris and washed twice with culture medium. The cells were then cultured with culture medium in an incubator with 5% CO2 and 97% humidity at 37ºC [18].

Characterization of neuronal-like lineage cells

Cell morphology: Morphological changes of the living cells were observed in the culture flasks on days 5, 10, 15, 25, 35, 45, 55, 60 and 65 by light microscopy using a Nikon binocular inverted microscope (model TS100 F).

Gene expression of neural markers by RT-PCR

Total RNA was extracted (RNeasy mini Kit, Qiagen) from 100% confluent cells on days 10, 25, 35, 45, 55, and 65, and the purity of the RNA was confirmed by determining the 260/280 nm absorbance ratio (>1.8). For each sample 1 μg of total RNA was reversed-transcribed to cDNA at 37°C for 60 min using the first strand cDNA Synthesis Kit (Thermo). PCR amplification was performed with 3 μl cDNA using HotStarTaq® Master Mix PCR from Qiagen. The PCR products were separated by electrophoresis through a 1.5% agarose gel stained with ethidium bromide and visualized using an UV illuminator. The RTPCR was repeated in all samples three times.

Using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a endogenous control, the gene expression of the following neuronal and glial markers were observed: Neuron specific enolase (NSE), neurofilament L (NF-L), intermediate filament protein nestin, a microtubule element of tubulin protein (β-tubulin III), zinc finger protein 521 ZNF521, neurotrophic tyrosine kinase receptor 1 (NTRK1), midkine (MDK) - also known as neurite growth promoting factor 2 – (NEGF2), and as glial cell markers claudin 11 (CLDN11) and glial fibrillary acidic protein (GFAP). All the primer sequences were determined using established gene bank sequences of oligonucleotide primers as listed in Table 1.

Primers bp TA C 0 cyc.
Neuronal markers      
Neuron Specific Enolase (NSE)      
F.: 5`-CTGATGCTGGAGTTGGATGG-3 188 55 28
R.: 5’-CCATTGATCACGTTGAAGGC-3’      
Neurofilament light (NF-L):      
F.: 5`-TCCTACTACACCAGCCATGT-3` 284 55 30
R.: 5`-TCCCCAGCACCTTCAACTTT-3`      
Nestin (NES)      
F.: 5'-AGGATGTGGAGGTAGTGAGA-3’ 251 53 31
R.: 5′-TGGAGATCTCAGTGGCTCTT-3′      
β- tubulin III      
F.: 5'-AGTGATGAGCATGGCATCGA-3' 317 53 28
R.: 5'-AGGCAGTCGCAGTTTTCACA-3'      
Zinc finger protein (Zfp521)      
F.: 5`-GGTGAAACTTGATATCAATGGCC-3` 482 60 29
F.: 5`-GGTGAAACTTGATATCAATGGCC-3`      
Neurotrophic tyrosine kinase receptor type1 (NTRK1)      
F.: 5’-TCTTCACTGAGTTCCTGGAG-3’ 229 60 32
R.: 5’-TTCTCCACCGGGTCTCCAGA-3’      
Neurite growth promoting factor2 (NEGF2) (MDK)      
F.: 5'-CGACTGCAAGTACAAGTTTGAGAAC-3' 110 53 30
R.: 5'-TCTCCTGGCACTGAGCATTG-3'      
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)      
F : 5`-GGG CTG CTT TTA ACT CTG CT-3` 702 55 28
R.: 5`-TGG CAG GTT TTT CTA GAC GG-3`      
Glial markers      
Claudin 11 (CLDN 11)      
F.: 5'-TGACCTGCGGCTACACCATCC-3' 412 58 28
R.: 5'-AGGCACAGCACAGCACCAATCC-3'      
Glial fibrillary acidic protein (GFAP)      
F.: 5’-CCTCTCCCTGGCTCGAATG-3’ 161 55 33
R.:5’-GGAAGCGAACCTTCTCGATGTA-3’      

Table 1: Oligonucleotide primers used for PCR.

Protein expression of neural markers by immunocytochemistry (ICC)

ICC was performed on days 35, 45 for identifying the expression of marker proteins. Cells (1×104 per ml) were cultured on sterile glass cover slips. The confluent cells were washed and fixed in 2% (v/v) paraformaldehyde for 4 hrs. The fixed cells were washed 3 times with DPBS in 5 min interval, then permeabilized in 0.2% Triton X-100 in DPBS for 10 min, washed again 3 times with DPBS, thereafter blocked for 1 hr in blocking buffer containing 1% bovine serum albumin (BSA) in DPBS, and after washing again incubated overnight with primary antibodies (1:200 in blocking buffer). Subsequently the cells were washed again 3 times and incubated for 2 hrs in fluoroisothiocyanate (FITC) conjugated secondary antibodies (1:200 in blocking buffer), afterwards washed, mounted with aqueous mounting medium, and visualized under a fluorescent microscope (Nikon, Japan). The primary antibodies and fluoroisothiocyanate (FITC) conjugated secondary antibodies are listed in Table 2. All antibodies used for immunocytochemistry were from Santacruz Biotechnology, Inc. Switzerland.

Protein Primary Antibody Secondary Antibody
Neuron specific enolase (NSE) Mouse Monoclonal Antibody Goat Anti-Mouse IgGFITC
Neurofilament light (NF-L) Mouse Monoclonal Antibody Goat Anti- Mouse IgGFITC
Growth associated protein 43 (GAP43) Goat Polyclonal Antibody Donkey Anti-Goat IgGFITC
Oligodendrocyte transcription factor2 (Oligo2) Goat Polyclonal Antibody Donkey Anti-Goat IgGFITC
CD13 (aminopeptidase N) Goat Polyclonal Antibody Donkey Anti-Goat IgGFITC
Synaptosome associated protein 25 (SNAP25) Goat Polyclonal Antibody Donkey Anti-Goat IgGFITC
Syntabulin Goat Polyclonal Antibody Donkey Anti-Goat IgGFITC
Glyceraldehyde-3-phosphate dehydrogenese) GAPDH Mouse Monoclonal Antibody Goat Anti-Mouse IgG
- FITC

Table 2: Antibodies used for immunocytochemistry.

Using ICC the early neural progenitor cells were examined for neuron specific enolase (NSE), neurofilament-L (NF-L), growth associated protein 43 (GAP43), CD13, synaptosomal associated protein (SNAP25), Syntabulin, and for glial cells oligodendrocytes transcription factor (Olig2). GAPDH was determined as endogenous control.

Results

Morphological changes

After 5 days in culture ADSCs began already to convert from fibroblastic- to neuronal-like morphology changing from elongated spindle like cells to cells with multiple small neurite-like processes. After 10 days cell types progressively adopted neuronal morphological characteristics: Slowly cytoplasm formed a contracted body and began to extend peripherally. On day 15 cytoplasm had retracted towards the nucleus and formed a contracted multipolar cell body with typical neuronal-like phenotype. During further growth the cells exhibit glial cells, formation of axons, and dendrite like cytoplasmic projections as sign of neuronal-like lineage cells. After 35 days cell bodies exposed multiple cell processes and elongated dendrites exhibiting a neuronal appearance and interlinking with other cells (Figure 1). Gene expression of neural and glial markers by RT-PCR.

Figure 1: Proceeding differentiation of ADSCs to neuronal-like lineage cells: Light microscopic appearance from day 5 till day 65. 1a: Glial-like cells appearance on day 5 (arrow) and continuous differentiation on day 10 and day 15. 1b. The development of glial and neuronal cells on day 25, 35 and 45. 1c. Continuous growth of neuronal-like cells and interlinking of cells.

Neuronal markers

Neuron-specific enolase (NSE), β-tubulin lll and midkine (MDK) were expressed strongly from day 10 to 65. Also the transcription factor ZNF521 could be detected strongly over the whole period. NF-L was found clearly till day 35 and declined to moderate expression after 6 weeks. NTRK1 was expressed clearly on day 10 and faded away to light appearance till day 65. Nestin showed only moderate to light expression from day10 to 65. We used GAPDH as endogenous control (Figure 2a and 2b).

Figure 2: Ge ne expressions of differentiated neuronal- and glial-like cells day 10, 25, 35, 45, 55, 65. 2a. Neuron specific genes expression of NSE, NF-L and β-tubulin lll. 2b. Neural precursors Nestin, transcription factor ZNF521, neurotrophic tyrosine kinase receptor1 NTRK1, neurite growth promoting factor 2 MDK, and control endogenous gene GAPDH. 2c. Glial marker claudin 11 and GFAP.

Glial markers

Claudin 11 was moderately expressed on day 10 and strongly from day 25 to 65. The expression of GFAP was moderate from day 10 till 35 and light only from day 45 till 65 (

Protein expression of neural and glial markers by ICC).

Neuronal markers: Cytoplasmic distribution of enolase (NSE), NFL, and GAP43 were revealed on day 35 and 45 (Figure 3a). Neuronal markers associated with the synaptic transmission CD13, syntabulin and SNAP25 were also expressed on day 35 and 45 (Figure 3b). The oligodendrocytes transcription factor 2 (Olig2) was expressed on day 35 and 45. GAPDH as endogenous control showed also positive expression on day 35 and 45 (Figure 3c).

Figure 3: Immuno-cytochemical staining of neuronal- and glial- like proteins on days 35 and 45. 3a. NSE, NF-L and GAP43. 3b. CD13, SNAP25 and syntabulin. 3c. Olig2 and GAPDH as control.

Discussion

The in-vitro differentiation of MSCs to specific lineage cells is a promising approach to gene therapy for tissue regeneration. Adipose tissue has proven to be an attractive source for generating pluripotent stromal cells [19,20]. After several studies demonstrated potentially positive effects of MSCs on the repair of nerve- and brain- damages many attempts were tried to differentiate early neural progenitor cells from BMSCs or ADSCs using “specific” neuronal induction protocols [9-11]. MSCs developed in different induction media neuronal- and glial-like appearances and gene- as well as protein- expression of typical neural markers.

However Lu et al. and Neuhuber et al. [14,15] demonstrated in their studies that chemical neuronal induction might be an artifact of cell stress due to toxic chemicals like β-mercaptoethanol (β-ME), dimethylsulfoxide (DMSO) or butylated hydroxyanisole (BHA) by adopting a neuron-like appearance due to the disruption of cytoskeleton. The exposure of neuronal-like phenotypes is thus the result of cell stress by toxic chemicals and not indicating true differentiation by reprogramming [21-23]. Moreover the basal mRNA expression pattern of undifferentiated human MSCs includes also characteristics of neuronal lineage cells and neuronal related mRNAs belong to the 50 most abundant gene expressions [24]. This hallmarks a weak point in almost all the studies differentiating MSCs into neuronal lineage cells.

Our study explores the differentiation potential of human ADSCs into neuronal lineage-cells without using any of the published induction protocols. To get homogeneous donor samples white fat out of the infrapatellar Hoffa-pad from 10 patients were selected and cultured over 65 days. As criteria for differentiation into neuronal-like lineage cells the morphological appearance and the gene- and proteinexpression of neuronal and glial markers were determined.

The cells exposed already after 5 days in culture medium with the conversion of elongated spindle-like cells to neuronal-like cells with multiple small neurite-like processes. On day 15 multipolar cell bodies with typical neuronal-like phenotype were developed. With some delay also glial-like cells could be observed and till day 65 the full appearance of neuronal-like interconnected cells was exhibited.

The neuronal and glial lineage cells observed in our investigation showed similar morphological characteristics as the neuronal-like cells in other studies differentiated from ADSCs using induction protocols or from embryonic stem cells.

Strong gene expression was exposed from day 10 to 65 of the following neuronal markers: NSE (neuron specific enolase), β-tubulin III (a key cytoskeletal and microtubule element protein - almost exclusively neuron specific), MDK (neurite growth promoting factor 2), and ZNF521 (the transcription factor Zinc finger nuclear protein considered to be for driving the intrinsic neural differentiation into neural progenitors). Neurofilament-L (NF-L), (which is implicated in synaptic plasticity) and neural precursors protein nestin were expressed moderately. NTRK1, neurotrophic tyrosine kinase receptor gene was expressed clearly only on day10 and declined and faded away till day 65.

The glial markers Claudin 11 (oligodendrocyte specific protein- OSP) was expressed clearly from day 10 till day 65 the GAFP expression was moderate from day 10 to 35 and light only till day 65.

The protein expression detected on the 5th and 6th week after culture by ICC was positive for NSE, NF-L, growth associated protein 43 (GAP 43), CD13 (identical to aminopeptidase N and involved in the synaptic membrane of the central nervous system), SNAP25 (synaptosome associated protein 25), and syntabulin (a microtubuleassociated protein implicated in syntaxin transport in neurons). As glial marker oligodendrocytes transcription factor 2 (Olig2) was expressed on days 35 and 45.

According to our results ADSCs contain a subset of cells, which can differentiate into neuronal lineage-cells expressing typical neuronal and glial markers at the posttranscriptional as well as posttranslational level. The spectrum of the exposed markers has a comparable validity as the neural markers selected in studies differentiating putative neurogenic cells from ADSCs using any induction protocol.

This study revealed typical indicators for the differentiation of ADSCs into neuronal-like lineage cells and shows furthermore that the use of so called specific induction protocols is not necessary for the differentiation of MSCs into early neural progenitors. Whether this process is a true reprogramming differentiation after gradually progressive restriction in the developmental potential must be evaluated by further studies. Moreover the question remained whether these differentiated neuronal-like cells can produce mature functional neurons with proven action potentials and synaptic transmitter release when given in a nerve or glial tissue environment.

Conclusion

A subset of cells within ADSCs can differentiate into neuronal lineage-cells without using a specific induction and express typical neuronal and glial markers at the post transcriptional and as well as posttranslational level. These markers are exposed in a comparable spectrum as markers selected in studies differentiating putative neurogenic cells from ADSCs using induction protocols.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this article.

Acknowledgement

This study was funded by Department of Science and Technology (DST) under Ministry of Science and Technology, a grant (SR/WOS-A/LS-193/2012). This Project in MIOT Institute of Research (MIR) is encouraged by MIOT International.

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