In culture medium, MSCs formed a monolayer of adherent cells and appeared as long, spindle-shaped fibroblastic cells. Capacity for chondrogenic, adipogenic, and osteogenic differentiation was confirmed with the use of a Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN). Cell surface antigens on the cells were analyzed by fluorescence-activated cell sorting (FACS), and we confirmed that the cells were positive for CD29, CD44, CD73, CD90, CD105, CD166, and MHC-DR but negative for CD14, CD34, and Flk-1, as described previously [30] (link).
Human mesenchymal stem cell functional identification kit
Manufactured by R&D Systems
Sourced in United States
The Human Mesenchymal Stem Cell Functional Identification Kit is a laboratory tool designed to characterize and identify human mesenchymal stem cells. The kit provides reagents and protocols to assess the functional properties of these cells.
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Lab products found in correlation
Alizarin red solution, by Merck Group (2 mentions)
Alizarin red s staining, by Merck Group (2 mentions)
Oil red staining, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Oil red o staining, by Merck Group (1 mentions)
Aggrecan, by R&D Systems (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
α mem, by R&D Systems (1 mentions)
Safranin o, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Alexa fluor 594, by Jackson ImmunoResearch (1 mentions)
Cloning cylinder, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Stemflow human msc analysis kit, by BD (1 mentions)
Cd90 fitc, by BD (1 mentions)
Cd105 pe, by BD (1 mentions)
Alcian blue, by Merck Group (1 mentions)
15 ml conical tube, by Corning (1 mentions)
Cd34 pe cy7, by BD (1 mentions)
30 protocols using human mesenchymal stem cell functional identification kit
1
Characterization of Mesenchymal Stem Cells
2
Multilineage Differentiation of ADSCs
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ADSCs and ADSCKs were tested for their ability to differentiate into adipocytes, chondrocytes and osteocytes using Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems), according to the manufacturer instructions. Adipogenic differentiation was performed starting from 2.1×104 cells/cm2 in SCM up to confluence, when SCM was replaced with Adipogenic Differentiation Medium (and changed every 3–4 days). After 21 days, cells were fixed with 4% paraformaldehyde for 1 hour and visualized by Oil Red O Staining (Sigma Aldrich).
Chondrogenic differentiation was performed with 2.5×105 cells seeded in 15 ml conical tubes in Chondrogenic Differentiation Medium (replaced every 2–3 days); after 21 days, chondrocyte pellet was fixed with 4% paraformaldehyde for 20 min and immunostained for aggrecan (R&D System).
Osteogenic differentiation was done starting form 4.2×103 cells/cm2 in SCM up to 70–80% confluence, when SCM was replaced with Osteogenic Differentiation Medium (changed every 3–4 days). After 21 days, cells were fixed 10 min with 70% ethanol and processed for Alizarian Red Staining (Sigma Aldrich). Images were obtained at 20X magnification, using Nikon Eclipse TE300 equipped with the Axiovision device camera (Zeiss Instr., Oberkochen, Germany). Images were processed using Axiovision release 4.6.3 (Zeiss Instr., Oberkochen, Germany).
Chondrogenic differentiation was performed with 2.5×105 cells seeded in 15 ml conical tubes in Chondrogenic Differentiation Medium (replaced every 2–3 days); after 21 days, chondrocyte pellet was fixed with 4% paraformaldehyde for 20 min and immunostained for aggrecan (R&D System).
Osteogenic differentiation was done starting form 4.2×103 cells/cm2 in SCM up to 70–80% confluence, when SCM was replaced with Osteogenic Differentiation Medium (changed every 3–4 days). After 21 days, cells were fixed 10 min with 70% ethanol and processed for Alizarian Red Staining (Sigma Aldrich). Images were obtained at 20X magnification, using Nikon Eclipse TE300 equipped with the Axiovision device camera (Zeiss Instr., Oberkochen, Germany). Images were processed using Axiovision release 4.6.3 (Zeiss Instr., Oberkochen, Germany).
3
Adipogenic Differentiation of Cultured ASCs
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The adipogenic, chondrogenic, and osteogenic differentiation of cultured ASCs was achieved using the Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Inc., Minneapolis, MN, USA), which contains specially formulated media supplements and a panel of antibodies to define the mature phenotypes of adipocytes, chondrocytes, and osteocytes, as previously reported [27 (link)]. In particular, for adipogenic differentiation, cells were resuspended in αMEM (Gibco) supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine and seeded on coverslips onto 24-well plates, at a density of approximately 3.7 × 104 cells/well. Medium was replaced every 2-3 days until 100% confluency was reached, then cells were incubated with adipogenic differentiation medium (ADM, αMEM supplemented with a solution containing hydrocortisone, isobutylmethylxanthine, and indomethacin; R&D Systems), to induce adipogenesis. In KGF-treated cells, ADM was supplemented with 1 ng/ml KGF (Upstate Biotechnology, Lake Placid, NY). The ADM or ADM + KGF was replaced every 3-4 days. The appearance of lipid vacuoles was monitored by microscopic examination.
4
Multipotent Stem Cell Differentiation
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To prove the multipotent properties, MSC were cultured with specific media supplements inducing differentiation into adipogenic, chondrogenic and osteogenic lineages. (Human Mesenchymal Stem Cell Functional Identification Kit; R&D Biosystems). After three weeks of differentiation, cells were fixed in 4% (w/v) paraformaldehyde and stained with Oil Red O (Sigma-Aldrich), Alizarin Red solution (Sigma-Aldrich) or Safranin O, respectively.
For MDC, the ability to differentiate into myotubes was assessed. Briefly, cells were cultured in myogenic differentiating medium (mDM) composed of DMEM with 4% HS and 10 μg/mL of insulin. Additionally, immunocytochemical stainings against desmin and sarcomeric myosin were performed. For this procedure, fixed cells were permeabilized with ice-cold methanol for 20 min. The non-specific binding of antibodies was blocked by 30 min incubation in a solution of 1% bovine serum albumin and 5% normal donkey serum in PBS. Primary monoclonal mouse anti-desmin antibody (clone D33, Dako, 1:30) or monoclonal mouse anti-sarcomeric myosin (clone MF20, Developmental Studies Hybridoma Bank, 1:30) were used (overnight, 4°C). After washing, the cells were incubated with donkey anti-mouse secondary antibody conjugated with Alexa-Fluor® 594 (Jackson ImmunoResearch Europe, 1:100, 60 min, RT).
For MDC, the ability to differentiate into myotubes was assessed. Briefly, cells were cultured in myogenic differentiating medium (mDM) composed of DMEM with 4% HS and 10 μg/mL of insulin. Additionally, immunocytochemical stainings against desmin and sarcomeric myosin were performed. For this procedure, fixed cells were permeabilized with ice-cold methanol for 20 min. The non-specific binding of antibodies was blocked by 30 min incubation in a solution of 1% bovine serum albumin and 5% normal donkey serum in PBS. Primary monoclonal mouse anti-desmin antibody (clone D33, Dako, 1:30) or monoclonal mouse anti-sarcomeric myosin (clone MF20, Developmental Studies Hybridoma Bank, 1:30) were used (overnight, 4°C). After washing, the cells were incubated with donkey anti-mouse secondary antibody conjugated with Alexa-Fluor® 594 (Jackson ImmunoResearch Europe, 1:100, 60 min, RT).
5
Isolation and Characterization of Mesenchymal Stem Cells from LO-EPC Cultures
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Colonies of MSC were identified in some LO-EPC preparations after 2 weeks in culture. Spindle-shaped MSC colonies were visually distinct from LO-EPC and were isolated using cloning cylinders (Sigma) and cultured separately in MSC culture medium, consisting of Dulbecco's modified Eagle's medium (DMEM) with 10% Hyclone fetal calf serum.
MSC differentiation and functional characterization were performed using a Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems) according to the manufacturer's instructions.
Oil Red O staining was performed by fixing cells in 10% formalin for 10 min at room temperature. After washing three times with distilled water, the cells were incubated with 60% isopropanol for 2 min and left to dry at room temperature. Cells were incubated in 60% Oil Red O solution for 5 min at room temperature. Excess stain was removed by washing with distilled water.
Alizarin red staining was performed by fixing cells with 70% ethanol for 1 h on ice. After washing with distilled water, cells were incubated in 2% alizarin red S solution for 5 min, followed by multiple washes with distilled water.
MSC differentiation and functional characterization were performed using a Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems) according to the manufacturer's instructions.
Oil Red O staining was performed by fixing cells in 10% formalin for 10 min at room temperature. After washing three times with distilled water, the cells were incubated with 60% isopropanol for 2 min and left to dry at room temperature. Cells were incubated in 60% Oil Red O solution for 5 min at room temperature. Excess stain was removed by washing with distilled water.
Alizarin red staining was performed by fixing cells with 70% ethanol for 1 h on ice. After washing with distilled water, cells were incubated in 2% alizarin red S solution for 5 min, followed by multiple washes with distilled water.
6
Multipotent Differentiation of Adipose-Derived Stem Cells
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Different human ASC lines were prepared from the fat of patients as described (Uckay, I., et al., J Stem Cell Res Ther, 2019. 9). Cells were cultured in Dulbecco's Modified Eagle Medium DMEM (4.5 g/L glucose, L‐Glutamine) supplemented with 10% of human platelet lysate (Stemulate, Cook Regentek), 1% penicillin and streptomycin (Thermo Fisher) at 37°C and under 5% CO2. This study was conducted according to the approval by local ethical committee of the University Hospitals of Geneva, Switzerland (2020–01102 and NAC 14–183). Written informed consent was obtained from each individual. The multipotent differentiation into adipocytes, osteocytes and chondrocytes was performed on a monolayer of ASC directly derived from ASC spheroids by using the Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems) according to the supplier's instructions.
7
Multilineage Differentiation Potential of MSCs
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The adipogenic, osteogenic and chondrogenic differentiation potentials of the HUMSCs and DPSCs were determined by incubating the cells in differentiation media using a Human Mesenchymal Stem Cell Functional Identification kit (R&D Systems, Inc, Minneapolis, MN, USA).
8
Multilineage Differentiation of EnSCs
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EnSCs were cultured and induced with human mesenchymal stem cell functional identification kit (R&D System, Minneapolis, Minnesota, USA) as instructed by the manufacturer. Cells were then fixed in 4 % formaldehyde. For osteogenic marker, cells were stained with Alizarin red for 5 min. For chondrogenic differentiation, cells were stained with Alcian blue for 30 min. For adipogenic differentiation, cells were stained with Oil Red O for 30 min [19 ].
9
Differentiation Potential of P4 MSCs
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P4 MSCs were assessed using the Human Mesenchymal Stem Cell Functional Identification Kit (R&D System) following the product instruction. After 21 days, cells were fixed and stained with FABP-4 antibody to identify adipocytes and osteocalcin antibody to identify osteocytes.
10
Multilineage Stem Cell Differentiation
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hMSC differentiation studies were conducted using the R&D Systems human mesenchymal stem cell functional identification kit according to the manufacturer’s directions using fatty acid binding protein 4 (FABP4), osteocalcin, and aggrecan as markers of adipocyte, osteoblast, and chondrocyte differentiation, respectively. Adipogenesis was further evaluated using oil red O staining [16 (link)]. Images were transmitted to a pathologist in blinded manner. Positive and negative cells were quantified in Image J; a minimum of 99 cells were counted per cohort.
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