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&#39;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.

Fluorescence-Activated Single-Cell Sorting of Human Mesenchymal Stem Cells

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+and+impact+of+mesenchymal+stem+cell+heterogeneity:+new+insights+and+emerging+tools+for+single+cell+analysis.">On the origin and impact of mesenchymal stem cell heterogeneity: new insights and emerging tools for single cell analysis.</a> European Cells &amp; Materials. 34, 217-231 (2017).</li><li>Dominici, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. 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M., Kameishi, S., Grainger, D. W., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+to+address+mesenchymal+stem/stromal+cell+heterogeneity+in+immunomodulatory+profiles+to+improve+cell-based+therapies.">Strategies to address mesenchymal stem/stromal cell heterogeneity in immunomodulatory profiles to improve cell-based therapies.</a> Acta Biomaterialia. 133, 114-125 (2021).</li><li>Yang, Y. K., Ogando, C. R., Wang See, C., Chang, T. Y., Barabino, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+phenotype+and+differentiation+potential+of+human+mesenchymal+stem+cells+aging+in+vitro.">Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro.</a> Stem Cell Research &amp; Therapy. 9 (1), 131 (2018).</li><li>Costa, L. 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The authors declare that there is no conflict of interest regarding the publication of this paper.

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...

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 architecture of MSCs2,4.
Immunophenotyping of cells has been a common application of flow cytometry for the identification of stem cells and this was&#160;utilized by the International Society for&#160;Cell and Gene Therapy (ISCT) in 2006 to prescribe a list of minimal criteria to identify cells as MSCs. It stated that along with plastic adherence and the ability to differentiate into three lineages (osteogenic, chondrogenic, and adipogenic) in vitro, &#8805;95% of the cell population must express CD105, CD73, CD90, and these cells must lack the expression (&#8804;2% positive) of CD34, CD45, CD11b, CD14, and HLA-DR, as measured by flow cytometry5. Although the MSCs were defined by a set of biomarkers under the minimal criteria of the ISCT, their immune properties could not be benchmarked with these biomarkers and there was a need for more beyond these criteria to make cross-study comparisons and clonal variations easier to quantify2.
Despite the guidelines set by ISCT, extensive research on MSCs has shown that heterogeneity exists in this population, which could arise due to a multitude of factors, mainly due to the ubiquitous nature of heterogeneity that arises between MSC donors6, tissue sources7, individual cells within a clonal population8, and culture conditions2,9,10. Characterization and purification of these primary cells from a variety of tissue sources to ensure quality and cell fate are key steps in their production. The need to understand the displayed variations amongst the population requires an efficient method to resolve it into subpopulations that can be divided and collected separately11. Single-cell level analyses help overcome the challenges of cell-cell variation, reduce biological noise arising from a heterogeneous population, and offer the ability to investigate and characterize rare cells12.
Based on the purpose and chosen parameters, several methods can be employed to sort and enrich the selected populations. Cell-sorting techniques can comprise both bulk sorting and single-cell sorting methods. While bulk sorting can enrich target populations through Magnetic-activated cell sorting (MACS)13, fractionation14, and elutriation15, single-cell sorting can enrich more homogeneous populations by means of fluorescence-activated cell sorting (FACS)11. A comparative analyses&#160;of each of these methods with its own set of advantages and disadvantages is highlighted in Table 1.
Table 1: Comparative analyses of different techniques:&#160;MACS, Fractionation, Elutriation, and FACS highlighting the differences in their principle and the advantages and disadvantages of choosing a particular technique over another. Abbreviations: MACS = Magnetic-activated cell sorting; FACS = Fluorescence-activated cell sorting. <a href="https://www.jove.com/files/ftp_upload/65723/65723_Table_1_.xlsx" target="_blank">Please click here to download this Table.</a>
Since the advent of the technique, single-cell flow cytometry has played a major role in enumeration16, detection, and characterization of a specific cell population in a heterogeneous sample17. Hewitt et al. in 2006 laid the foundation of automated cell sorting methodology to enhance the isolation of homogenous pools of differentiated human embryonic stem cells (hESCs)18. Single-cell sorting enriched the population of GFP-transduced hESCs facilitating the isolation of genetically modified clones, which opened a new dimension in clinical research. To improve the sort efficiency, two approaches have generally been taken; either the collection media of the sorted populations are modified to sustain viability and proliferation of post-sorted cells19 or the cell-sorting algorithm/software is appropriately modified12.
With the advancement of technology, commercial flow cytometers and cell sorters have been able to help address challenges that were met while aseptically sorting fragile and rare cell populations, especially stem cells of different origins.&#160;One of the major challenges of stem cell biologists has been the clonal isolation of human pluripotent stem cells following transfection protocols required in gene editing studies19. This was addressed by sorting single cells into 96-well plates that were coated with Mouse Embryonic Fibroblasts (MEFs) along with supplements and commercial small molecule ROCK inhibitors. However, cell isolation strategies could be largely refined with the use of index sorting, a feature of the sorting algorithm that identifies the immunophenotype of individual cells sorted12. This refined modality in single-cell sorting helped not only in enhancing sort efficiency for stem cells, especially with regard to rare hematopoietic stem cell populations, but also efficiently linked single-cell clones to their downstream functional assays20.
This paper focuses on single-cell sorting of immunophenotyped&#160;stem cells from human&#160;exfoliated deciduous teeth (SHEDs) for the enrichment of sub-populations to study their functional differentiation capacities. Using a combination of two MSC-positive markers, CD90 and CD73, and a negative hematopoietic marker CD45, the MSCs were immunophenotyped and the dim and null expressors were identified. Based on their immunophenotype the subpopulations were identified as pure MSCs, single positive and double negative populations. They were sorted using the single-cell sort mode to obtain pure and enriched subpopulations for further functional studies to identify whether the differential expression of markers was an artifact of in vitro culture conditions or whether it has any effect on the functional properties as well. Cells that were not homogeneous expressors of the &#34;positive MSC markers&#34; were sorted to study their functional properties.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alcian Blue Stain</td>
			<td>HiMedia</td>
			<td>CCK029-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100x)&#160;</td>
			<td>Gibco by ThermoFisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>BD CompBead Plus Anti-Mouse Ig, &#954;/Negative Control (BSA) Compensation Plus (7.5 &#181;m) Particles Set</td>
			<td>BD Biosciences</td>
			<td>560497</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS Accudrop Beads&#160;</td>
			<td>BD Biosciences</td>
			<td>345249</td>
			<td>Used to set up the Laser delay when the sort module opens.</td>
		</tr>
		<tr>
			<td>BD FACS Aria Fusion Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>---</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS Diva 9.4</td>
			<td>BD Biosciences</td>
			<td>---</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS Sheath Fluid</td>
			<td>BD Biosciences</td>
			<td>342003</td>
			<td>Used as sheath fluid for both analysis and sorting experiments in the BD FACSAria Fusion</td>
		</tr>
		<tr>
			<td>BD FACSDiva CS&#38;T Research Beads</td>
			<td>BD Biosciences</td>
			<td>655050</td>
			<td>Used for Instrument configuration depending on the nozzle size.</td>
		</tr>
		<tr>
			<td>BD Horizon V450 Mouse Anti-Human CD44</td>
			<td>BD Biosciences</td>
			<td>561292</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Horizon V450 Mouse IgG2b, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>560374</td>
			<td>CD44-V450 isotype</td>
		</tr>
		<tr>
			<td>BD Pharmingen APC Mouse Anti-Human CD105</td>
			<td>BD Biosciences</td>
			<td>562408</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen APC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>555751</td>
			<td>CD105-APC isotype</td>
		</tr>
		<tr>
			<td>BD Pharmingen DAPI Solution</td>
			<td>BD Biosciences</td>
			<td>564907</td>
			<td>DAPI Stock solution of 1 mg/mL</td>
		</tr>
		<tr>
			<td>BD Pharmingen FITC Mouse Anti-Human CD90</td>
			<td>BD Biosciences</td>
			<td>555595</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen FITC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>555748</td>
			<td>CD90-FITC isotype</td>
		</tr>
		<tr>
			<td>BD Pharmingen PE Mouse Anti-Human CD45</td>
			<td>BD Biosciences</td>
			<td>555483</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen PE Mouse IgG1, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>555749</td>
			<td>CD45-PE isotype</td>
		</tr>
		<tr>
			<td>BD Pharmingen PerCP-Cy 5.5 Mouse Anti-Human CD73</td>
			<td>BD Biosciences</td>
			<td>561260</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen PerCP-Cy 5.5 Mouse IgG1, &#954; Isotype Control</td>
			<td>BD Biosciences</td>
			<td>550795</td>
			<td>CD73-PerCP-Cy 5.5 isotype</td>
		</tr>
		<tr>
			<td>BD Pharmingen Purified Mouse Anti-Vimentin</td>
			<td>BD Biosciences</td>
			<td>550513</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Hi-Media&#160;</td>
			<td>TC548-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Nice chemical pvt ltd&#160;</td>
			<td>C33809</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma-aldrich&#160;&#160;</td>
			<td>D5652-50L</td>
			<td>dPBS used for culture work and maintenance.&#160;</td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>---</td>
			<td>---</td>
			<td>Used for general sterlization.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>A-21422</td>
			<td></td>
		</tr>
		<tr>
			<td>KO-DMEM</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>10829018</td>
			<td>Basal medium for undifferentiated hESCs, used in the preparation of culture media</td>
		</tr>
		<tr>
			<td>L-Glutamine 200mM (100x)</td>
			<td>Gibco by ThermoFisher</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol, for Molecular Biology&#160;</td>
			<td>Hi-Media&#160;</td>
			<td>MB113</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil red O</td>
			<td>HiMedia&#160;</td>
			<td>CCK013-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>loba chemie</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (100x)</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>15140- 122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin (ActinGreen 488 ReadyProbes reagent)</td>
			<td>Invitrogen&#160;</td>
			<td>R37110</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Nitrate</td>
			<td>HiMedia&#160;</td>
			<td>MB156-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Thiosulphate pentahydrate</td>
			<td>Chemport</td>
			<td>10102-17-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sphero Rainbow Fluorescent Particles, 3.0 - 3.4 &#181;m</td>
			<td>BD Biosciences</td>
			<td>556291</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining buffer&#160;</td>
			<td>Prepared in MIRM&#160;</td>
			<td>----</td>
			<td>It was prepared using 2% FBS in PBS&#160;</td>
		</tr>
		<tr>
			<td>StemPro Adipogenesis Differentiation Basal Media&#160;</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>A10410-01</td>
			<td>Basal media for Adipogenic media</td>
		</tr>
		<tr>
			<td>StemPro Adipogenesis Supplement</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>A10065-01</td>
			<td>Induction media for Adipogenic media</td>
		</tr>
		<tr>
			<td>StemPro Chondrogenesis Supplement</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>A10064-01</td>
			<td>Induction media for Chondrogenic media</td>
		</tr>
		<tr>
			<td>StemPro Osteogenesis Supplement</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>A10066-01</td>
			<td>Induction media for Osteoogenic media</td>
		</tr>
		<tr>
			<td>StemPro Osteogenesis/Chondrogenesis Differentiation Basal Media&#160;</td>
			<td>Gibco by ThermoFisher&#160;</td>
			<td>A10069-01</td>
			<td>Basal media for both Ostegenic and Chondrogenic media</td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Hi-Media&#160;</td>
			<td>MB031</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue&#160;</td>
			<td>Gibco by life technologies&#160;</td>
			<td>15250-061</td>
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		</tr>
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			<td>Trypsin - EDTA Solution 1x</td>
			<td>Hi-media&#160;</td>
			<td>TCL049</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20&#160;</td>
			<td>MERCK&#160;</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
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</table>

single-cell sorting of immunophenotyped mesenchymal stem cells from human exfoliated deciduous teeth

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&#160;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&#160;stem cells from&#160;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 &#34;bright,&#34; positive-expressors of MSCs&#160;using multiparametric&#160;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.

Author Spotlight: Importance of Single Cell Sorting in Isolating Purified Populations of Mesenchymal Stem Cells 

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

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...

Watch this Scientific Journal Video about Importance of Single Cell Sorting in Isolating Purified Populations of Mesenchymal Stem Cells at JoVE.com

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 ...

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JoVE Journal

Biology

1.1K Views.  Manipal Academy of Higher Education, Manipal. 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.