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Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth

Published: November 10th, 2023



1Manipal Institute of Regenerative Medicine, Bengaluru, Manipal Academy of Higher Education, Manipal, 2HIV-AIDS Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR)
* These authors contributed equally

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based approach.

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and are known for their immunomodulatory and anti-inflammatory properties. The use of these stem cells is a boon to the field of regenerative biology, but at the same time, a bane to regenerative medicine and therapeutics owing to the multiple cellular ambiguities associated with them. These ambiguities may arise from the diversity in the source of these stem cells and from their in vitro growth conditions, both of which reflect upon their functional heterogeneity.

This warrants methodologies to provide purified, homogeneous populations of MSCs for therapeutic applications. Advances in the field of flow cytometry have enabled the detection of single-cell populations using a multiparametric approach. This protocol outlines a way to identify and purify stem cells from human exfoliated deciduous teeth (SHEDs) through fluorescence-assisted single-cell sorting. Simultaneous expression of surface markers, namely, CD90-fluorescein isothiocyanate (FITC), CD73-peridinin-chlorophyll-protein (PerCP-Cy5.5), CD105-allophycocyanin (APC), and CD44-V450, identified the "bright," positive-expressors of MSCs using multiparametric flow cytometry. However, a significant drop was observed in percentages of quadruple expressors of these positive markers from passage 7 onwards to the later passages.

The immunophenotyped subpopulations were sorted using the single-cell sort mode where only two positive and one negative marker constituted the inclusion criteria. This methodology ensured the cell viability of the sorted populations and maintained cell proliferation post sorting. The downstream application for such sorting can be used to evaluate lineage-specific differentiation for the gated subpopulations. This approach can be applied to other single-cell systems to improve isolation conditions and for acquiring multiple cell surface marker information.

Mesenchymal stem cells (MSCs) can be regarded as a scalable source of cells suitable for cell-based therapies and may be considered a gold standard system in regenerative medicine. These cells can be isolated from a variety of sources in the body with different tissue origins1. Depending on their source tissue, each type of MSC displays an ambiguous in vitro behavior2. This is well observed in their morphological and functional properties3. Multiple studies have shown intra-clonal variation in dimensions, including adult tissue differentiation, genomic state, and metabolic and cellular ar....

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Ethics approval and consent to participate: Human exfoliated deciduous dental pulp samples were received after obtaining informed consent and full ethical approval by Sri Rajiv Gandhi Dental College and Hospital (SRGCDS) Oral and Maxillofacial Department, Bengaluru, in accordance with the standards established by the Hospital Ethical Clearance Committee, SRGCDS. Following which isolation, culture, maintenance, and application of SHEDs were approved by and in compliance with the guidelines recommended by the Institut.......

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The SHEDs were characterized with standard immunofluorescence assays showing the expression of vimentin (red, type III intermediate filaments), actin filaments (Alexa fluor 488 Phalloidin Probes), and nuclei stained with DAPI (Figure 1A). To estimate their proliferative and colony-forming capacities, standard short-term cell growth assays were performed. A 14.3-fold increase in proliferation rate from day 2 to day 8 has been shown in Figure 1B. The clonogenic pr.......

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In the field of tissue engineering and regenerative medicine, among the postnatal sources, oral tissue-derived MSCs have attracted profound interest because of their minimal ethical obligations and notable multilineage differentiation potential21. Dental pulp stem cells (DPSCs) from the impacted third molar and SHEDs have garnered the most attention among dental MSCs for their therapeutic potential in neurodegenerative and traumatic diseases22. The protocol described in thi.......

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We thank the Flow Cell Facility at Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, India, for the use of the flow cytometry core facility. The cryo-sectioning of the pellet culture of differentiated cells was performed at Neuberg Anand Reference Laboratory, Bengaluru, India. This work was supported by UC's intramural funding from the Manipal Academy of Higher Education (MAHE), India. AG is grateful for the support of the Dr. T. M. A. Pai Scholarship from MAHE.


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Name Company Catalog Number Comments
Alcian Blue Stain HiMedia CCK029-1KT
Antibiotic-Antimycotic (100x)  Gibco by ThermoFisher 15240062
BD CompBead Plus Anti-Mouse Ig, κ/Negative Control (BSA) Compensation Plus (7.5 µm) Particles Set BD Biosciences 560497
BD FACS Accudrop Beads  BD Biosciences 345249 Used to set up the Laser delay when the sort module opens.
BD FACS Aria Fusion Flow cytometer BD Biosciences ---
BD FACS Diva 9.4 BD Biosciences ---
BD FACS Sheath Fluid BD Biosciences 342003 Used as sheath fluid for both analysis and sorting experiments in the BD FACSAria Fusion
BD FACSDiva CS&T Research Beads BD Biosciences 655050 Used for Instrument configuration depending on the nozzle size.
BD Horizon V450 Mouse Anti-Human CD44 BD Biosciences 561292
BD Horizon V450 Mouse IgG2b, κ Isotype Control BD Biosciences 560374 CD44-V450 isotype
BD Pharmingen APC Mouse Anti-Human CD105 BD Biosciences 562408
BD Pharmingen APC Mouse IgG1, κ Isotype Control BD Biosciences 555751 CD105-APC isotype
BD Pharmingen DAPI Solution BD Biosciences 564907 DAPI Stock solution of 1 mg/mL
BD Pharmingen FITC Mouse Anti-Human CD90 BD Biosciences 555595
BD Pharmingen FITC Mouse IgG1, κ Isotype Control BD Biosciences 555748 CD90-FITC isotype
BD Pharmingen PE Mouse Anti-Human CD45 BD Biosciences 555483
BD Pharmingen PE Mouse IgG1, κ Isotype Control BD Biosciences 555749 CD45-PE isotype
BD Pharmingen PerCP-Cy 5.5 Mouse Anti-Human CD73 BD Biosciences 561260
BD Pharmingen PerCP-Cy 5.5 Mouse IgG1, κ Isotype Control BD Biosciences 550795 CD73-PerCP-Cy 5.5 isotype
BD Pharmingen Purified Mouse Anti-Vimentin BD Biosciences 550513
Bovine serum albumin Hi-Media  TC548-5G
Crystal violet Nice chemical pvt ltd  C33809
Dulbecco's Phosphate Buffered Saline Sigma-aldrich   D5652-50L dPBS used for culture work and maintenance. 
Ethanol  --- --- Used for general sterlization.
Fetal Bovine Serum  Gibco by ThermoFisher  10270-106
Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 ThermoFisher Scientific  A-21422
KO-DMEM Gibco by ThermoFisher  10829018 Basal medium for undifferentiated hESCs, used in the preparation of culture media
L-Glutamine 200mM (100x) Gibco by ThermoFisher 25030-081
Methanol, for Molecular Biology  Hi-Media  MB113
Oil red O HiMedia  CCK013-1KT
Paraformaldehyde  loba chemie 30525-89-4
Penicillin Streptomycin (100x) Gibco by ThermoFisher  15140- 122
Phalloidin (ActinGreen 488 ReadyProbes reagent) Invitrogen  R37110
Silver Nitrate HiMedia  MB156-25G
Sodium Thiosulphate pentahydrate Chemport 10102-17-7
Sphero Rainbow Fluorescent Particles, 3.0 - 3.4 µm BD Biosciences 556291
Staining buffer  Prepared in MIRM  ---- It was prepared using 2% FBS in PBS 
StemPro Adipogenesis Differentiation Basal Media  Gibco by ThermoFisher  A10410-01 Basal media for Adipogenic media
StemPro Adipogenesis Supplement Gibco by ThermoFisher  A10065-01 Induction media for Adipogenic media
StemPro Chondrogenesis Supplement Gibco by ThermoFisher  A10064-01 Induction media for Chondrogenic media
StemPro Osteogenesis Supplement Gibco by ThermoFisher  A10066-01 Induction media for Osteoogenic media
StemPro Osteogenesis/Chondrogenesis Differentiation Basal Media  Gibco by ThermoFisher  A10069-01 Basal media for both Ostegenic and Chondrogenic media
Triton-X-100 Hi-Media  MB031
Trypan Blue  Gibco by life technologies  15250-061
Trypsin - EDTA Solution 1x Hi-media  TCL049
Tween-20  MERCK  9005-64-5

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