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><li>Kobolak, J., Dinnyes, A., Memic, A., Khademhosseini, A., Mobasheri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells%3A+Identification%2C+phenotypic+characterization%2C+biological+properties+and+potential+for+regenerative+medicine+through+biomaterial+micro-engineering+of+their+niche%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Mesenchymal stem cells: Identification, phenotypic characterization, biological properties and potential for regenerative medicine through biomaterial micro-engineering of their niche.</a> Methods. 99, 62-68 (2016).</li>
<li>Wilson, A., Hodgson-Garms, M., Frith, J. E., Genever, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplicity+of+mesenchymal+stromal+cells%3A+finding+the+right+route+to+therapy%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">Multiplicity of mesenchymal stromal cells: finding the right route to therapy.</a> Frontiers in Immunology. 10, 1112(2019).</li>
<li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+the+biological+characteristics+of+human+mesenchymal+stem+cells+derived+from+exfoliated+deciduous+teeth%2C+bone+marrow%2C+gingival+tissue%2C+and+umbilical+cord%5BTitle%5D)+AND+%22Molecular+Medicine+Reports%22%5BJournal%5D)">Comparison of the biological characteristics of human mesenchymal stem cells derived from exfoliated deciduous teeth, bone marrow, gingival tissue, and umbilical cord.</a> Molecular Medicine Reports. 18 (6), 4969-4977 (2018).</li>
<li>McLeod, C. M., Mauck, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+origin+and+impact+of+mesenchymal+stem+cell+heterogeneity%3A+new+insights+and+emerging+tools+for+single+cell+analysis%5BTitle%5D)+AND+%22European+Cells+%26+Materials%22%5BJournal%5D)">On the origin and impact of mesenchymal stem cell heterogeneity: new insights and emerging tools for single cell analysis.</a> European Cells & Materials. 34, 217-231 (2017).</li>
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<li>Wang, J., Liao, L., Wang, S., Tan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+therapy+with+autologous+mesenchymal+stem+cells-how+the+disease+process+impacts+clinical+considerations%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">Cell therapy with autologous mesenchymal stem cells-how the disease process impacts clinical considerations.</a> Cytotherapy. 15 (8), 893-904 (2013).</li>
<li>Kern, S., Eichler, H., Stoeve, J., Kluter, H., Bieback, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+analysis+of+mesenchymal+stem+cells+from+bone+marrow%2C+umbilical+cord+blood%2C+or+adipose+tissue%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue.</a> Stem Cells. 24 (5), 1294-1301 (2006).</li>
<li>Dunn, C. M., Kameishi, S., Grainger, D. W., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strategies+to+address+mesenchymal+stem%2Fstromal+cell+heterogeneity+in+immunomodulatory+profiles+to+improve+cell-based+therapies%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">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/pubmed?term=((Changes+in+phenotype+and+differentiation+potential+of+human+mesenchymal+stem+cells+aging+in+vitro%5BTitle%5D)+AND+%22Stem+Cell+Research+%26+Therapy%22%5BJournal%5D)">Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro.</a> Stem Cell Research & Therapy. 9 (1), 131(2018).</li>
<li>Costa, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+heterogeneity+of+mesenchymal+stem+cells+from+natural+niches+to+culture+conditions%3A+implications+for+further+clinical+uses%5BTitle%5D)+AND+%22Cellular+and+Molecular+Life+Sciences%22%5BJournal%5D)">Functional heterogeneity of mesenchymal stem cells from natural niches to culture conditions: implications for further clinical uses.</a> Cellular and Molecular Life Sciences. 78 (2), 447-467 (2021).</li>
<li>Bonner, W. A., Hulett, H. R., Sweet, R. G., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+activated+cell+sorting%5BTitle%5D)+AND+%22Review+of+Scientific+Instruments%22%5BJournal%5D)">Fluorescence activated cell sorting.</a> Review of Scientific Instruments. 43 (3), 404-409 (1972).</li>
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<li>Liao, X., Makris, M., Luo, X. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence-activated+cell+sorting+for+purification+of+plasmacytoid+dendritic+cells+from+the+mouse+bone+marrow%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Fluorescence-activated cell sorting for purification of plasmacytoid dendritic cells from the mouse bone marrow.</a> Journal of Visualized Experiments. (117), (2016).</li>
<li>Roda, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+stem+cell+tag-less+sorting+method%5BTitle%5D)+AND+%22Stem+Cell+Reviews+and+Reports%22%5BJournal%5D)">A novel stem cell tag-less sorting method.</a> Stem Cell Reviews and Reports. 5 (4), 420-427 (2009).</li>
<li>Hall, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+isolation+of+small+CD44-negative+mesenchymal+stem%2Fprogenitor+cells+from+human+bone+marrow+using+elutriation+and+polychromatic+flow+cytometry%5BTitle%5D)+AND+%22Stem+Cells+Translational+Medicine%22%5BJournal%5D)">Identification and isolation of small CD44-negative mesenchymal stem/progenitor cells from human bone marrow using elutriation and polychromatic flow cytometry.</a> Stem Cells Translational Medicine. 2 (8), 567-578 (2013).</li>
<li>Eggleton, M. J., Sharp, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Platelet+counting+using+the+Coulter+electronic+counter%5BTitle%5D)+AND+%22Journal+of+Clinical+Pathology%22%5BJournal%5D)">Platelet counting using the Coulter electronic counter.</a> Journal of Clinical Pathology. 16 (2), 164-167 (1963).</li>
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<li>Hewitt, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence-activated+single+cell+sorting+of+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Cloning+and+Stem+Cells%22%5BJournal%5D)">Fluorescence-activated single cell sorting of human embryonic stem cells.</a> Cloning and Stem Cells. 8 (3), 225-234 (2006).</li>
<li>Singh, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+efficient+protocol+for+single-cell+cloning+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Frontiers+in+Cell+and+Developmental+Biology%22%5BJournal%5D)">An efficient protocol for single-cell cloning human pluripotent stem cells.</a> Frontiers in Cell and Developmental Biology. 7, 11(2019).</li>
<li>Wilson, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combined+single-cell+functional+and+gene+expression+analysis+resolves+heterogeneity+within+stem+cell+populations%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Combined single-cell functional and gene expression analysis resolves heterogeneity within stem cell populations.</a> Cell Stem Cell. 16 (6), 712-724 (2015).</li>
<li>Zhou, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oral+mesenchymal+stem%2Fprogenitor+cells%3A+the+immunomodulatory+masters%5BTitle%5D)+AND+%22Stem+Cells+Inernational%22%5BJournal%5D)">Oral mesenchymal stem/progenitor cells: the immunomodulatory masters.</a> Stem Cells Inernational. 2020, 1327405(2020).</li>
<li>Fawzy El-Sayed, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+mesenchymal+stem+cells+explored+in+the+dental+field%5BTitle%5D)+AND+%22Advances+in+Biochemical+Engineering%2FBiotechnology%22%5BJournal%5D)">Adult mesenchymal stem cells explored in the dental field.</a> Advances in Biochemical Engineering/Biotechnology. 130, 89-103 (2013).</li>
<li>Hardy, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+networks+in+single+perivascular+cells+sorted+from+human+adipose+tissue+reveal+a+hierarchy+of+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Transcriptional networks in single perivascular cells sorted from human adipose tissue reveal a hierarchy of mesenchymal stem cells.</a> Stem Cells. 35 (5), 1273-1289 (2017).</li>
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</ol>

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><tbody><tr><td>Alcian Blue Stain</td><td>HiMedia</td><td>CCK029-1KT</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x)&amp;nbsp;</td><td>Gibco by ThermoFisher</td><td>15240062</td><td></td></tr><tr><td>BD CompBead Plus Anti-Mouse Ig, &amp;kappa;/Negative Control (BSA) Compensation Plus (7.5 &amp;micro;m) Particles Set</td><td>BD Biosciences</td><td>560497</td><td></td></tr><tr><td>BD FACS Accudrop Beads&amp;nbsp;</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&amp;amp;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, &amp;kappa; 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, &amp;kappa; 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, &amp;kappa; 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, &amp;kappa; 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, &amp;kappa; 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&amp;nbsp;</td><td>TC548-5G</td><td></td></tr><tr><td>Crystal violet</td><td>Nice chemical pvt ltd&amp;nbsp;</td><td>C33809</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma-aldrich&amp;nbsp;&amp;nbsp;</td><td>D5652-50L</td><td>dPBS used for culture work and maintenance.&amp;nbsp;</td></tr><tr><td>Ethanol&amp;nbsp;</td><td>---</td><td>---</td><td>Used for general sterlization.</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco by ThermoFisher&amp;nbsp;</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&amp;nbsp;</td><td>A-21422</td><td></td></tr><tr><td>KO-DMEM</td><td>Gibco by ThermoFisher&amp;nbsp;</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&amp;nbsp;</td><td>Hi-Media&amp;nbsp;</td><td>MB113</td><td></td></tr><tr><td>Oil red O</td><td>HiMedia&amp;nbsp;</td><td>CCK013-1KT</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>loba chemie</td><td>30525-89-4</td><td></td></tr><tr><td>Penicillin Streptomycin (100x)</td><td>Gibco by ThermoFisher&amp;nbsp;</td><td>15140- 122</td><td></td></tr><tr><td>Phalloidin (ActinGreen 488 ReadyProbes reagent)</td><td>Invitrogen&amp;nbsp;</td><td>R37110</td><td></td></tr><tr><td>Silver Nitrate</td><td>HiMedia&amp;nbsp;</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 &amp;micro;m</td><td>BD Biosciences</td><td>556291</td><td></td></tr><tr><td>Staining buffer&amp;nbsp;</td><td>Prepared in MIRM&amp;nbsp;</td><td>----</td><td>It was prepared using 2% FBS in PBS&amp;nbsp;</td></tr><tr><td>StemPro Adipogenesis Differentiation Basal Media&amp;nbsp;</td><td>Gibco by ThermoFisher&amp;nbsp;</td><td>A10410-01</td><td>Basal media for Adipogenic media</td></tr><tr><td>StemPro Adipogenesis Supplement</td><td>Gibco by ThermoFisher&amp;nbsp;</td><td>A10065-01</td><td>Induction media for Adipogenic media</td></tr><tr><td>StemPro Chondrogenesis Supplement</td><td>Gibco by ThermoFisher&amp;nbsp;</td><td>A10064-01</td><td>Induction media for Chondrogenic media</td></tr><tr><td>StemPro Osteogenesis Supplement</td><td>Gibco by ThermoFisher&amp;nbsp;</td><td>A10066-01</td><td>Induction media for Osteoogenic media</td></tr><tr><td>StemPro Osteogenesis/Chondrogenesis Differentiation Basal Media&amp;nbsp;</td><td>Gibco by ThermoFisher&amp;nbsp;</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&amp;nbsp;</td><td>MB031</td><td></td></tr><tr><td>Trypan Blue&amp;nbsp;</td><td>Gibco by life technologies&amp;nbsp;</td><td>15250-061</td><td></td></tr><tr><td>Trypsin - EDTA Solution 1x</td><td>Hi-media&amp;nbsp;</td><td>TCL049</td><td></td></tr><tr><td>Tween-20&amp;nbsp;</td><td>MERCK&amp;nbsp;</td><td>9005-64-5</td><td></td></tr></tbody></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.

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

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.

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 ...

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Biology

1.8K Views.  Manipal Academy of Higher Education, Manipal. Dit protocol beschrijft een cruciale methode voor het nauwkeurig karakteriseren en zuiveren van menselijke mesenchymale stamcellen op één enkel platform, waardoor efficiënte stroomafwaartse toepassingen zoals stamceltransplantatie mogelijk worden. Het is met name belangrijk voor klinische laboratoria die moleculaire en cytogenetische tests verbeteren door middel van nauwkeurige sortering van gemanipuleerde of natuurlijke stamcellen in één cel. De veelgebruikte methoden voor het isoleren ...

Video: Auteur in de schijnwerpers: Belang van sortering van enkele cellen bij het isoleren van gezuiverde populaties van mesenchymale stamcellen 

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Genetics

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Medicine

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

Immunology and Infection

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

Developmental Biology

Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which are present in both deciduous and permanent teeth, have emerged as a vital stem cell source due to their accessibility, adaptability, and innate differentiation capabilities. DPSCs offer a readily available and abundant reservoir of mesenchymal stem cells, showcasing impressive versatility and potential, particularly for regenerative purposes. Despite their promise, the main hurdle lies in effectively isolating and characterizing DPSCs, given their representation as a minute fraction within dental pulp cells. Equally crucial is the proper preservation of this invaluable cellular resource. The two predominant methods for DPSC isolation are enzymatic digestion (ED) and outgrowth from tissue explants (OG), often referred to as spontaneous growth. This protocol concentrates primarily on the enzymatic digestion approach for DPSC isolation, intricately detailing the steps encompassing extraction, in-lab processing, and cell preservation. Beyond extraction and preservation, the protocol delves into the differentiation prowess of DPSCs. Specifically, it outlines the procedures employed to induce these stem cells to differentiate into adipocytes, osteoblasts, and chondrocytes, showcasing their multipotent attributes. Subsequent utilization of colorimetric staining techniques facilitates accurate visualization and confirmation of successful differentiation, thereby validating the caliber and functionality of the isolated DPSCs. This comprehensive protocol functions as a blueprint encompassing the entire spectrum of dental pulp stem cell extraction, cultivation, preservation, and characterization. It underscores the substantial potential harbored by DPSCs, propelling forward stem cell exploration and holding promise for future regenerative and therapeutic breakthroughs.

This protocol emphasizes the extraction, culture, and preservation of multipotent stem cells from dental pulp through enzymatic digestion. Additionally, it demonstrates their potential to differentiate into osteoblasts, adipocytes, and chondrocytes, highlighting the importance of precision and consistency in the process.

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which ...

Bioengineering

Niche-Based Isolation of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.

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

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