In vitro differentiation of neural cells from human adipose tissue derived stromal cells
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.232326
Source of Support: None, Conflict of Interest: None
Keywords: Adipose tissue, mesenchymal stromal cells, neural cells, neurological disorders
All neurons are involved in a network that is important for specific physiological functions. Neurological disorders represent an irreversible loss of neurons, and human neurodegenerative disorders are caused by the loss or damage of neurons in the spinal cord or brain. Regenerating a “new” nerve tissue, which can restore its function, is the only hope in such a case. At present, where unavailability of effective clinical therapies for neurodegenerative disorders and spinal cord injuries are the main concerns, stem cells and neural stem cells (NSCs) are of particular translational interest. The roots of stem cells discovery belongs to the early 1960s; however, recent developments in stem cell-based therapies have opened up the gateway for neural cells generation and/or regeneration, in vitro as well as in vivo. Infusion or implantation of in-vitro generated neural cells in animal models of neurodegenerative disorders appears to be a promising therapeutic intervention. Clinical improvement as well as extension of life in these animal models have been demonstrated. The previous idea that the adult central nervous system is not capable of neurogenesis has been nullified with the discovery of this fact and subsequent research on neural cells.
With the use of rapidly advancing technology and research in the field of stem cell biology, damaged or lost neurons can be repaired/regenerated/replaced by transplantation or infusion of in-vitro expanded neural cells. For the patients with neurodegenerative disorders and/or chronically-injured spinal cord, in-vitro generated neural cells would be able to offer an effective cell-based treatment to restore the lost function.
Below-mentioned procedures/methodologies were carried out after obtaining approval of the Institutional Review Board (IRB). The participants' informed written consents were taken for the same.
Formulation of proliferation medium for the generation and proliferation of mesenchymal stromal cells
The proliferation medium was prepared to collect the resected adipose tissue, as well as for its further culture and generation of mesenchymal stromal cells (MSCs). The proliferation medium used here was formulated using minimum essential medium-alpha modified [α-MEM] (Sigma, USA), 5 ng basic-human fibroblast growth factor (B-hFGF) (Sigma, USA), 20% human albumin (Reliance Life Sciences, India), and 1% sodium pyruvate [Hi-media, India]. To control/avoid microbial contamination, a solution of penicillin and streptomycin (10 mg/l) [Hi-media, India] with cefotaxime (1 mg/ml) [Lupin, India] and fluconazol (10 mg/ml) [Medimark Biotech, India] was used for administration of antibiotics.
To generate and harvest mesenchymal stromal cells
Using local anesthesia, approximately 10–15 g of volunteer human participant's/donor's adipose tissue was surgically resected from the anterior abdominal wall. This resection was carried out by the surgeon by placing a small incision on the left lateral side, a little below the umbilicus, which was followed by taking of sutures at the incision site. The proliferation medium was used to collect the resected adipose tissue.
In the collected adipose tissue, an enzyme (to dissolve the extracellular matrix of adipose tissue) collagenase type-I (1 mg/1 ml) was added and the adipose tissue was slowly minced into fine pieces under a sterile condition. The minced adipose tissue was incubated by placing the content at 37°C in an incubator equipped with a shaker at 5 G for 60 minutes. After incubation for 60 min, the content was subjected to centrifugation at 65 G for 8 minutes. After centrifugation, the supernatant and pellet were both cultured separately in separate tissue culture dishes by adding the proliferation medium specific for generation of MSCs. The supernatant and pellets were cultured into the cell + tissue culture dishes (Sarstedt, Germany) of 100 cm 2 and 25 cm 2, respectively. Culture was carried out in the CO2 incubator at 37°C temperature, having 5% CO2, for 9–10 days. The medium from the tissue culture dishes was replenished using freshly prepared proliferation medium every other day.
To harvest and study in vitro generated mesenchymal stromal cells
After 9–10 days of culture, phosphate buffered saline [PBS, 1 N] (Hi-Media, India) was used to wash the cells, after which they were harvested using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution (Hi Media, India). The cells were washed again with 1 N PBS. For checking the viability of the harvested cells, supravital staining using trypan blue was carried out. Sterility was checked microscopically as well as using an automated machine Bactec (USA). Cell-counting of harvested MSCs was carried out using modified Neubauer chamber. As one of the confirmatory tests for harvested MSCs, phenotypic expression for flowcytometer markers, i.e., cluster of differentiation (CD) 45 (Per CP), CD90 (FITC), and CD73 (PE) (Beckton Dickinson, USA) were checked for their negative and positive expressions, respectively. For the study of morphological features, harvested MSCs were subjected for asssessment using the hematoxylin and eosin (H and E) stain followed by microscopic examination of the stained slides.
Differentiation of neural cells
For the differentiation of neural cells, MSCs were further cultured with the addition of neural differentiation medium. This specific medium was formulated using neurobasal medium (Invitrogen, Germany), Dulbecco's modified Eagle's medium: F-12 (DMEM: F-12) [Sigma, USA], nonessential amino acids (Sigma, USA), growth factors such as epidermal growth factor (EGF) (10 ng/ml) [Sigma, USA], brain derived neurotrophic factor (BDNF) (0.01 mg/ml) (Sigma, USA), glial derived neurotrophic factor (GDNF) [0.01 mg/ml] (Sigma, USA), cyclic-adenosine monophosphate (c-AMP) [60 μg/ml] (Sigma, USA), L-glutamine [0.5 mM] (Sigma, USA), laminin (5 μg/ml) [Sigma, USA], and N2 and B27 serum supplements (Sigma, USA). Antibiotics mentioned previously (for the formulation of proliferation medium) were also added to the differentiation medium.
Harvested MSCs were further kept in the abovementioned neural differentiation culture medium for 5–7 days. At the end of 5–7 days, for the culture in neural differentiation medium, cells were washed with PBS (1 N) [Hi-media, India] for 2–3 times and isolated. The isolated cells were then subjected to testing of their sterility, viability, and cell counts. The harvested cells were also subjected to immunofluorescence (IF) analysis for the assessment of neural cell transcription factor proteins such as orthodenticle homolog 2 (OTX-2) [Sigma, USA], beta III-tubulin [Sigma, USA], glial fibrillary acidic protein (GFAP) [Sigma, USA] and synaptophysin-β2 [Sigma, USA]. For immunofluorosce (IF) analysis, secondary antibodies used were goat anti-mouse IgG coupled with Alexa flour 488 (Invitrogen, Germany) diluted in PBS (1 N) [1:1000].
To study negative controls, MSCs (harvested on the initial 9–10th day of culture) were subjected to further culture without the use of neural differentiation medium and were allowed to continue to grow with the addition of a proliferation medium (a proliferation medium was used instead of a differentiation medium). Cell culture was carried out with the same culture conditions of 5% CO2 at 90% humidity for the same time period (as that required for the differentiation of neural cells, i.e., 5–7 days). At the end of the culture, cells were isolated and were subjected to qualitative, quantitative, and characterization assays, as was the protocol followed for neural cells.
Statistical analysis was performed using the Statistical Package for the Social Sciences version 12. Data were expressed as mean ± standard deviation (SD (with a range from minimum-maximum) for continuous variables. Continuous variables were compared using Wilcoxon signed rank test. P < 0.05 was considered to be statistically significant.
From 50 different volunteer donors, a total of 50 neural cell-lines were differentiated using in-vitro derived MSCs from adipose tissue.
Quantitative and qualitative analysis
Mesenchymal stromal cells
The MSC mean cell quantum was 3 ml and the mean cell count was 3.29 ± 1.6 × 103/μl (1.1–8 × 103/μl). Flowcytometric analysis revealed mean CD45-/90+ cells, 41.28 ± 21.03% (range: 22.1–87.67%) and mean CD45-/73+ cells, 14.59 ± 12.74% (range: 11–60.66%) [Figure 1]a. Morphological analysis by H and E stain showed cells with dendrites of variable sizes. A large basophilic nucleus with very finely distinct margins was observed, which was found surrounded by an eosinophilic/amphophilic cytoplasm [Figure 1]b.
The mean quantum of in-vitro generated neural cells was 5.4 ± 6.9 ml (1.1–8.6 ml) with the mean cell count of 5.27 ± 2.65 × 103/μl (1.1–12.3 × 103/μl).
Characterization assay for neural cells
IF staining of neural cells revealed significant expression of neural transcription factor proteins OTX-2, GFAP, beta III-tubulin, and synaptophysin-β2, which provided evidence for the presence of differentiated neural cells. All in-vitro generated neural cells showed the presence of neural transcriptional factor protein markers [Figure 2].
In contrast to the MSCs cultured further using the specific abovementioned differentiation medium, all the negative controls cultured without the use of the differentiation medium failed to show the expression of neural transcriptional factor protein markers OTX-2, GFAP, beta III-tubulin, and synaptophysin-β2.
Since the pioneering experiments of Spemann and Mangold which showed that the vertebrate nervous system can be “induced” by signals that emanate from a region of the embryo known as the “organizer,” formation of nervous system has been the center of attention for vertebrate embryologists. Clinical trials of transplantation using dopaminergic neurons recovered from human fetuses have been carried out, with the outcome indicating that it can replace endogenous degenerating dopamine neurons and can also provide some improvement in symptoms. However, the poor availability of tissues, ethical issues, and concerns with regard to the safety and quality control are the limitations for further studies in this area.
Since the discovery of stem cells, taking into account their promising role in clinical situations, a revolution in medical research and an alteration in the perception of the human body has occurred. Stem cells are basically nonspecific, undifferentiated, and unspecialized cells having significant differentiation and self-renewable capacity. Naturally occurring stem cells can be classified into embryonic stem cells, fetal stem cells, and adult stem cells. Adult stem cells are classical examples of multipotent cells and are defined by their tissue of origin. Multiple areas within the adult body, including bone marrow, brain, liver, and muscle, contain an endogenous adult stem cell population. The potential use of adult stem cells in autologous therapies, in which cells can be harvested and used within the same patient, is a key benefit of adult stem cells. This benefit eliminates the risks as well as ethical concerns associated with embryonic stem cells.
Stem cell research could lead to the development of radical new therapies for several neurodegenerative diseases that currently lack effective treatment. Over the past few years, there has been a continuous progress in developing approaches to generate the types of human-derived neurons and glial cells that are needed for cell replacement therapy based on the pathology in the respective diseases. Therapeutics using regenerative medicines may be an answer to neurological disorders and injuries, which are a major cause of disability worldwide. The main targets of stem cell therapies are developmental, degenerative, and traumatic neurological conditions in the spinal cord as well as the brain. In the field of neuroscience research, the successful differentiation of stem cells into neural cells has nullified the previous idea that the adult central nervous system (CNS) was not capable of neurogenesis. These neural cells are believed to have the potential to theoretically produce all cell types of CNS.,, The idea of using neural cells for the treatment of neurodegeneration as well as for chronic neurological injury is intriguing, as neural cells have been proven to give rise to glial-restricted and neuron-restricted precursors. Both of them can be induced to differentiate into astrocytes, oligodendrocytes, or neurons. Experimental data suggests that there may be improvement in functions by replacing the lost neurons and glial cells and by mediating in the process of remyelination, by the trophic actions, and by the modulation of inflammation by transplantation of stem cells or their derivatives in animal models of neurodegenerative diseases.,,
Stem cells have been used in many patients of various disorders since the last few years. Whether or not these stem cells can differentiate into functional neurons; and, whether or not they can provide trophic support to the injured neurons in patients with neurodegenerative disorders, were the key questions during the initial stages of experimentation in the field. Reynolds and Weiss were the first to isolate stem cells and neural progenitor cells from the adult strial tissue and subventricular zone of adult mice brain. Various tissue culture proliferation and/or differentiation media and relevant protocols are available for the study of functional characterization and progeny of neural cells using in vitro culture conditions since the discovery of neural cells. Neurosphere assay is such a culture system assay introduced in 1992, which has been widely used to differentiate, expand, isolate, and to quantify neural cell population. Various culture conditions and differentiation protocols till date have demonstrated efficiency of their own, suggesting a phenomenon of neural differentiation through different cellular signaling mechanisms.
In-vitro experiments have proven that, with the exposure of specific growth factors, stem cells and their progeny can be stimulated towards further proliferation. Among the varius types of stem cells, multipotent MSCs have been proven to be of significant application in the field of regenerative medicine and tissue engineering. MSCs are plastic-adherent and fibroblast-like cells. They can self-renew and have the differentiation potential of tissues from the mesodermic lineage. Clinical datausing in vitro and in vivo animal and human models have shown a broad field of application for MSCs. MSCs have been effective in preclinical central nervous system disease models. Differentiation of cells of nonmesenchymal origin, e.g., hepatocytes, neurons, and pancreatic islet cells, has been observed in vitro with MSC being their source, when specific culture conditions and stimuli were applied. Thus, the generation of neural cells using MSCs also have been suggested by a few studies.
Adipose tissue represents an abundant as well as a comparatively easily accessible source for the generation of MSCs because it is abundant in quantity and is an accessible source, which has the ability to differentiate along multiple lineage pathways. Information on the potency and biology of AD-MSCs has been obtained from various studies using in vitro as well as in vivo culture conditions. Stem cells derived from adipose tissue, i.e., MSCs, are capable of differentiating into bone, cartilage, fat, and muscle, similar to the phenomenon seen with bone marrow. Likewise, the capability of AD-MSC to adopt a neural-like phenotype makes them a promising source of neural cells for use in replacement therapy in the field of regenerative medicine.,
In our study, to achieve differentiation of neural cells using AD-MSC as a source, various culture conditions including serum-free differentiation medium has been used. Neural-differentiation medium comprising neurobasal medium had been used in combination with N2 and B27 serum supplements. This combination has been indicated to be helpful for the maintenance of brain regions' derived neurons, including those from the cortex, cerebellum, dentate gyrus, striatum, and substantia nigra. It also plays a significant role in supporting the growth of neural cell populations with no need of an astrocyte feeder layer or any other type of animal-origin feeder cell layer. The neurobasal medium was originally indicated as a supportive medium for primary neural cultures., Serum-free medium formulation used in our protocol with DMEM Ham's F12 media, supplies essential amino acids along with vitamins, inorganic salts, and some other nutrient materials necessary for cell growth. Addition of supplements in the form of nonessential amino acids to our differentiation medium plays a role in stimulating growth and prolonging the viability of cells in culture. EGF plays an important role in regulating cell growth, cell proliferation, their survival, as well as their further differentiation. C-AMP used in culture medium have been proven to induce MSCs into neural lineage. Neurotrophic factors such as GDNF and BDNF are growth factors that enhance the development of neural cells of the nervous system because they play a significant role in their survival and functionality. These growth factors enhance neural cells growth, differentiation, survival, and function in vitro. Laminin is a very active part of the basal lamina, which is of great biological importance. Laminin mainly influences cell differentiation, cellular phenotypic expression, and survival. Both in-vitro and in-vivo experiments have proven laminin as a major substrate along which the nerve axons grow.
Signal transduction pathways work to trigger the activation of neural transcription factors through which neural cells differentiation and maintenance occurs. Use of neural-differentiation medium initiates the activation and/or deactivation of various intracellular signaling pathways initiated from ligand molecules, which reaches to different molecular and cellular effectors, and from which gene expression modulation is accelerated, ultimately resulting in neural phenotypic adaption of MSCs. For regulation of expression of genes responsible for neural cell proliferation and multipotency, transcriptional factor proteins specific for neural differentiation, i.e., OTX-2, β3-Tubulin, GFAP, and synaptophysin-β2 are believed to play a significant role. Mainly, the protein kinase-A (PKA) pathway, mitogen-activated protein kinases (MAPKs) pathway, phospholipase-C (PLC) pathway, and phosphatidyl-inositol-3-kinase (PI3K) pathway are activated by the specific growth factors and ingredients used in the formulation of neural-differentiation medium, which results in the phosphorylation of neural transcriptional factor proteins. These activated transcriptional factor proteins in turn regulate the commitment, differentiation, and activation of neural lineage by regulating their genetic expression. They are also responsible for survival and synaptic function of neural-specific lineages., In embryonic development as well as in neural cell survival and neural plasticity, these pathways have been demonstrated to be of fundamental importance.,, With the application of various growth factors and specific culture conditions for culture, the cells adopted neural morphology and expression of markers specific for neural transcription factor proteins in our characterization assay, for in-vitro AD-MSC differentiated neural cells.
We opted for an in-vitro generation of neural cells because we believe that, for providing lasting and appropriate neurological recovery in neurodegenerative disorders and spinal cord injuries, neural cells are the potential and important candidates, with promising outcomes of research carried out in the field. Experimental data suggest the mechanisms of remyelination and trophic support to transplanted neural cells, wherein the spinal niche directs the transplanted cells to terminally differentiate into more numbers of neural cell types/lineages, making this treatment beneficial. However, further research is needed in the field before its clinical use. Adequate numbers of reports in the literature showing generation and/or therapeutic use of neural cells are lacking; we believe that stem cell-based therapy and gene therapy, particularly using neural cells, shall be the potential gold-mine of future research in the field of regenerative medicine treating neurodegenerative disorders and chronic spinal cord injury.
The authors are thankful to Mr Jignesh Patel for carrying out flowcytometer work. Mr B Patel, Mr J Chudasama and Mrs P N Bhavsar carried out the immunofluorescence staining.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2]