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ORIGINAL ARTICLE  
Year : 2011  |  Volume : 54  |  Issue : 3  |  Page : 501-508
Detection of embryonic stem cell markers in adult human adipose tissue-derived stem cells


Research, Training and Applications, MIOT Hospitals, 4/112, Mount Poonamallee Road, Manapakkam, Chennai, Tamil Nadu, India

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Date of Web Publication20-Sep-2011
 

   Abstract 

Background: Bone marrow transplantation is already an established therapy, which is now widely used in medicine to treat leukemia, lymphoma, and several inherited blood disorders. The culture of multilineage cells from easily available adipose tissue is another source of multipotent mesenchymal stem cells, and is referred to as adipose tissue-derived stem cells (ADSCs). While ADSCs are being used to treat various conditions, some lacuna exists regarding the specific proteins in these. It was therefore decided to analyze the specific proteins of embryonic cells in ADSCs. Aims: To analyze the specific protein of embryonic stem cells (ESCs) in ADSCs. Materials and Methods: Adult human adipose tissue-derived stem cells (ADSCs) were harvested from 13 patients after obtaining patients' consent. The specific markers of ESCs included surface proteins CD10, CD13, CD44, CD59, CD105, and CD166, and further nucleostemin,(NS) NANOG, peroxisome proliferator-activated receptor-gγ, collagen type 1 (Coll1), alkaline phosphate, (ALP) osteocalcin (OC), and core binding factor 1 (Cbfa1) were analyzed using by reverse transcription-polymerase chain reaction, (RT-PCR) immunofluorescence (IF), and western blot. Results: All the proteins were expressed distinctly, except CD13 and OC. CD13 was found individually with different expressions, and OC expression was discernable. Conclusions: Although the ESC with its proven self-renewal capacity and pluripotency seems appropriate for clinical use, the recent work on ADSCs suggests that these adult stem cells would be a valuable source for future biotechnology, especially since there is a relative ease of procurement.

Keywords: Adipose tissue-derived stem cells, immunofluorescence, stem cell markers, tissue engineering

How to cite this article:
Arumugam SB, Trentz OA, Arikketh D, Senthinathan V, Rosario B, Mohandas P. Detection of embryonic stem cell markers in adult human adipose tissue-derived stem cells. Indian J Pathol Microbiol 2011;54:501-8

How to cite this URL:
Arumugam SB, Trentz OA, Arikketh D, Senthinathan V, Rosario B, Mohandas P. Detection of embryonic stem cell markers in adult human adipose tissue-derived stem cells. Indian J Pathol Microbiol [serial online] 2011 [cited 2020 Nov 30];54:501-8. Available from: https://www.ijpmonline.org/text.asp?2011/54/3/501/85082



   Introduction Top


Research in stem cells began when the Canadian scientists in 1960s reported on the presence of self-renewing cells within the bone marrow of mice and postulated that these cells were regenerative stem cells. [1],[2] In the 1950s, experiments with bone marrow established the existence of stem cells, and late 1960s led to the development of bone marrow transplantation, a therapy now widely used in medicine to treat leukemia, lymphoma, and several inherited blood disorders. [3],[4]

A few years ago, Zuk et al. [5] described the culture of multilineage cells from adipose tissues as another source of multipotent mesenchymal stem cells. There are also many other sources to obtain adult stem cells such as bone marrow stem cells (BMSCs), skin, digestive epithelium, dental pulp, hair follicles, brain, amniotic fluid, placenta, etc. [6],[7] The proliferative ability and multilineage potential of easily available human adipose tissue is a promising approach for future tissue engineering. [8],[9],[10],[11] However, some lacuna still exists regarding the specific proteins in the adipose tissue-derived stem cells (ADSCs), which are responsible for proliferation. It was therefore decided to observe the development of stem cells from adipose tissue and observe their characteristics with special analysis of their specific proteins. Thus, the aim of this study was to detect specific proteins of embryonic stem cells (ESCs) in adult human ADSCs.


   Materials and Methods Top


ADSCs were harvested from Hoffa fat pads taken during knee arthroplasties. All patients gave their informed consent and the study was carried out according to existing ethical guidelines. Specific proteins of ESCs were identified in ADSCs from 13 patients (2 men and 11 women; mean age, 60.0 ± 8 years; range, 50-74 years).

Isolation and Culture of Adipose Tissue-derived Stem Cells

All tissues reached the laboratory in sterile saline within 30 min of harvesting and subjected to culture.

The fat tissues were washed with Dulbecco's phosphate-buffered saline (DPBS; Gibco, NY, USA) without calcium and magnesium to remove the blood. Following this, small vessels and fascia were separated from the fat tissue, inside the lamina flow bench [Figure 1]a. The isolated fat tissue was minced and put in a small sterile container or in a sterile 50-ml tube with 7-10 ml (depending upon the quantity of fat tissue) of 0.075% collagenase type 1 (Sigma, Buchs, Switzerland) dissolved in DPBS and digested at 37°C for 12 h in an incubator [Figure 1]b. The enzyme activity was neutralized with the culture medium [500 ml Dulbecco's modified Eagle's medium (DMEM) medium with 10% fetal calf serum (FCS), 60 mg/ml ascorbic phosphate, and 60 mg/ml antibiotic-antimycotic; Invitrogen] and centrifuged for 10 min at 1800 rpm. The sediment was resuspended in the culture medium and filtered through a 70 mm nylon mesh (BD Falcon, MD, USA) to remove cellular debris and washed twice with the culture medium. The counted cells were cultured in the culture medium in an incubator with 5% CO 2 and 97% humidity at 37°C.
Figure 1: (a) Adipose tissue from Hoff a's fat pad. (b) Adipose tissue after digestion with collagenase. (c) Adipose tissue, Oil red "O" staining, x10. (d) Adipose tissue, Oil red "O" staining, x20

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Characterization of Cells

The confluent cells were characterized by the determination of well-established stem cell markers of surface proteins (CD10, CD13, CD44, CD59, CD105, and CD166), and gene expression of nucleostemin (NS), NANOG, peroxisome proliferator-activated receptor gamma 2 (PPAR-g2), osteocalcin (OC), core binding factor alpha 1 (Cbfa1), alkaline phosphatase (ALP), and collagen type 1 (Coll 1), using reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence (IF), and western blot. Adherent cells from passage 0 and passage 1 (from day 7 till day 14) were used for RNA extraction.

Histology

Hematoxylin-eosin staining

Confluent cells were fixed with 2% paraformaldehyde and stained with Mayer's hematoxylin as standard method and mounted using DPX (di-n-butylphthalate in xylene).

Oil red "O" staining

The frozen adipose tissue was sectioned in 15 mm slices and mounted on slides. The slides were fixed for 5 min in 10% formalin and washed four times with distilled water. This was followed by incubation in 60% isopropanol for 5 min; the tissue was dried and later stained using the Oil red "O" working solution for 10-15 min. After rinsing with isopropanol to remove the Oil red "O" solution and finely counterstaining with Mayer's hematoxylin solution for 2 min, the slides were mounted and observed under a light microscope.

Alizarin red staining

After fixing with 2% paraformaldehyde and washing, the cells were stained with the 2% alizarin red solution for 2 min (2 g alizarin in 100 ml dH2 O, pH. 4.1-4.2). After 2 min, the cells were washed with dH 2 O, mounted, and viewed under the microscope.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction

RNA was isolated from 100% confluent cells with a Qiagen kit (Basel, Switzerland) as recommended, and reverse transcribed to cDNA, using Omniscript® reverse transcriptase, as described in the protocol. Each reaction was prepared in a total reaction mixture of 20 ml, containing 1 mg of total RNA, and was converted using reverse transcriptase at 37°C for 60 min. PCR amplification was performed with 3 ml RT product (cDNA), using HotStarTaq ® Master Mix PCR Kit from Qiagen (Basel, Switzerland) according to the instructions. Duplicate PCR reactions were also amplified for the housekeeping gene, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control (same amount of the RT product) for assessing PCR efficiency and for subsequent analysis by agarose gel electrophoresis. All the primer sequences were determined using established gene bank sequences' oligonucleotide primers as listed in [Table 1].
Table 1: Oligonucleotide primers for polymerase chain reaction

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Immunofluorescence

ADSCs were cultured on sterile glass cover slips (passage 1) and fixed for 20 min in 2% paraformaldehyde. The cells were washed three times in a washing buffer (DPBS; Gibco) and permeabilized with 0.2% Triton x 100 in DPBS. The cells were washed again three times and then blocked for 1 h in the blocking buffer containing 1% bovine serum albumin (BSA) in DPBS. They were washed three times in the washing buffer and incubated overnight with the primary antibody (Coll 1 mouse monoclonal antibody; Santacruz Biotechnology Inc.,). The cells were washed extensively with the washing buffer and incubated for 2 h in the fluoroisothiocyanate (FITC)-conjugated secondary antibody (goat anti-mouse IgG; Santacruz Biotechnology). To confirm the NS expression, cover slips were incubated overnight with the goat polyclonal primary antibody (Santacruz Biotechnology). After washing, the cells were incubated for 2 h in the FITC-conjugated secondary antibody (donkey anti-goat IgG; Santacruz Biotechnology). NANOG and CD166 were detected using the goat polyclonal primary antibody and incubated for 2 h in the FITC-conjugated donkey anti-goat IgG secondary antibody (Santacruz Biotechnology). After that, the cells were washed three times with the washing buffer, and mounted with the aqueous mounting medium.

For diaminobenzidine (DAB), staining we used the horseradish peroxidase (HRP)-conjugated secondary antibody from Sigma (Buchs, Switzerland). The cells were incubated with 0.5 ml of a DAB-enhanced liquid substrate system (30 ml DAB liquid chromogen solution + 1 ml of the DAB liquid buffer solution; Sigma), for 2 min in the dark at room temperature. The reaction was stopped by washing with water and mounted with the aqueous mounting medium.

Western Blot

Protein was extracted with the rapid immunoprecipitation assay buffer (RIPA; 50 mM Tris HCl, pH. 7.4, 150 mM NaCl, 2 mM EDTA, and 1% Nonidet P-4, and 0.1% SDS). After removing the culture medium, cells were washed three times with ice-cold DPBS, added 500 ml cold RIPA buffer, and kept in ice for 30 min with occasional swirling for uniform spreading. The cell lysate was collected using a cell scraper and transferred to a microcentrifuge tube and centrifuged at 3000 rpm for 30 min at 4°C to pellet the cell debris. The supernatant containing the protein was then transferred to a new tube and the protein concentration was determined using the Lowry method. [12] Western blot was performed for the proteins NS (goat polyclonal antibody; Santacruz Biotechnology.), and GAPDH (mouse monoclonal antibody; Santacruz Biotechnology) by the method of Towbin et al. [13] A total of 25 mg of boiled protein lysate was loaded onto NuPAGE 4-12% bis-Tris acrylamide gels (Invitrogen, Basel, Switzerland), electrophoresed, and transferred to nitrocellulose membranes. The membranes were blocked in DPBS with 5% skimmed milk powder and 0.1% Tween for 2 h at room temperature. After washing, they were incubated with a primary antibody diluted 1:400 in PBS with skimmed powdered milk and 0.1% Tween overnight with rocking at 4°C. The membranes were then incubated with the peroxidase-conjugated secondary antibody (NS: donkey anti-goat IgG-HRP, GAPDH: goat anti-mouse IgG-HRP; Santacruz Biotechnology), with skimmed milk powder and 0.1% Tween rocking for 2 h at room temperature. After washing with DPBS/0.1% Tween three times, the blots were developed using the ECL kit (BioVision Inc., USA).


   Results Top


Histology

The adipogenic nature of the adipose tissue was confirmed by the dark red staining of the lipid droplets by the Oil Red "O" solution in the adipose tissue sections [Figure 1]c and d. ADSCs expanded easily in vitro and exhibited a fibroblast-like morphology and with the number of passages, these cells developed a fattish configuration [Figure 2]a and b. Hematoxylin-eosin staining showed very clearly the nucleus [Figure 2]c and d. Oil red "O" staining of ADSCs after 10 days did not show any sign of fat tissue [Figure 2]e and alizarin red staining showed no sign of mineralization [Figure 2]f.
Figure 2: (a) ADSCs 10 days after culture x10. (b) ADSCs 21 days after culture, x10. (c) ADSCs, H-E staining after 10 days, x10. (d) ADSCs, H-E staining after 21 days, x10. (e) ADSCs Oil red "O" staining 10 days after culture, x10. (f) ADSCs alizarin staining 10 days after culture, x10

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Reverse Transcription-Polymerase Chain Reaction

The undifferentiated ADSCs were consistently positive for the surface proteins CD10, CD44, CD59, CD105, and CD166, except CD13, which could not depict the same in all 13 patients. These cells also revealed gene expression of NS, ALP, PPARg2 and GAPDH except Cbfa1. However, OC, the only osteoblast-specific gene, was also expressed sparsely along with no further verified genes [Figure 3]a, b and c.
Figure 3: (a)Expression of surface proteins: CD10 (cyc.28), CD 13 (cyc.30), CD44 (cyc.27), CD59 (cyc 28), CD105 (cyc27), and CD166 (cyc28).
Figure 3b: Expression of gene osteocalcin (OC cyc 28), nucleostemin (NS cyc 27), GAPDH (cyc 27), alkaline-phosphatase (ALP cyc 28), core binding factor (Cbfa1 cyc29) and peroxisome proliferator-activated receptor gamma (PPARg2 cyc 30).
Figure 3c: Osteocalcin gene expression of another 10 patients to confirm the results, and as control 1, 2, and 13 expression of OC in mature human osteoblasts (as positive control)


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Immunofluorescence

GAPDH and NANOG expressions could be distinguished very well in IF and also by DAB [Figure 4]a-d. However, the NS expression could be confirmed very clearly in the nucleoli of ADSCs and the same was also observed for Coll 1 in the cytoplasm [Figure 5]a and b. CD166 was conspicuous in IF and PPAR-g2 expression could be recognized very weakly by DAB [Figure 5]c and d.
Figure 4: (a) GAPDH as an endogenous control, IF x20. (b) GAPDH as endogenous control, DAB x20. (c) Transcription factor NANOG, IF x20. (d) Transcription factor NANOG, DAB x20

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Figure 5: (a) Nucleostemin expression 10 days after culture, IF x20. (b) Collagen type 1 expression 10 days after culture, IF x20. (c) CD166 expression 10 days after culture, IF x20. (d) PPAR gamma expression 10 days after culture, DAB x20

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Additionally, the expression of NS and GAPDH was confirmed by immunoblotting [Figure 6].
Figure 6: Western immunoblotting expression of nucleostemin and GAPDH 10 days after culture

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   Discussion Top


Many investigators have shown that adipose tissue is a rich source of autologous regenerative cells. [14],[15],[16],[17],[18] Adipose tissue-derived regenerative cells consist of adult stem cells, endothelial progenitor cells (blood vessel forming cells), and other growth factor-producing cells (tissue growth and repair promoting cells). The purpose of this study was to observe the development of stem cells from adipose tissue and their characterization for ESC-specific proteins. The surface proteins CD10, CD44, CD59, CD105, and CD166 were consistently positive except CD13 in these undifferentiated adipose tissue-derived cells of 13 patients [Figure 3]a. CD13 considered being expressed in mesenchymal stem cells, endothelial cells, and monocytes[19] showed individual different expressions.

Kafienah described in 2006 [20] NS as a new nucleolar protein and a marker of undifferentiated human adult bone marrow and ADSCs, which is involved in the regulation of proliferation of these cells. In this study, NS was also very clearly expressed by RT-PCR, western immunoblotting, and IF. Tsai and MacKay [21] described NS as a novel P53-binding protein localized in the nucleoli of stem cells and cancer cells, but absent in differentiated cells. Bernardi and Pandolfi [22] identified NS as a cDNA expressed in rat stem cells in the central nervous system. It entirely disappears at the start of differentiation, and the expression of NS is abruptly downregulated during differentiation. Prior to terminal cell division, it also participates in the control of stem cells and cancer cell proliferation. The exact function of NS is not yet revealed; it is believed to bind to the p53 protein and regulate the proliferation of stem cells and is markedly downregulated during cellular differentiation throughout the embryonic development and in adulthood.

PPAR-γ2, a ligand-activated transcription factor, exposed individual different expressions by RT-PCR. PPAR-γ2 is a key regulator of adipocyte differentiation in bone metabolism. [23],[24] This altered sequence of adipose gene expression in ADSCs may be due to a distinct developmental program characteristic of stem cells. Consistent with this, OC (the most osteoblast specific protein) expression, an established late marker of osteoblast differentiation, is also observed in ADSCs [Figure 3]b and c. Alternatively, the observed gene sequences may be due to the asynchronous development of cell subpopulations within the heterogenous ADSCs. [18] Cbfa1, [25] the transcription factor of OC, was also identifiable in ADSCs.

All these data support the use of adipose tissue as a potential source for multipotent cells and principally a suitable approach for future regenerative medicine, tissue engineering applications, and valuable resource in biotechnology. [26] Despite a lot of experimental evidence, convincing clinical data are still missing and future studies are necessary to establish the role of the long-term differentiation of specialized cells for regenerative medicine in vitro and in vivo.

 
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Correspondence Address:
Sarasa Bharati Arumugam
Research, Training and Applications, MIOT Hospitals, 4/112, Mount Poonamallee Road, Manapakkam, Chennai - 600 089, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.85082

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    Figures

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