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 architecture of MSCs2,4.
Immunophenotyping of cells has been a common application of flow cytometry for the identification of stem cells and this was utilized by the International Society for 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, ≥95% of the cell population must express CD105, CD73, CD90, and these cells must lack the expression (≤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 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: 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. Please click here to download this Table.
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. 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 stem cells from human 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 "positive MSC markers" were sorted to study their functional properties.
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 Institutional Committee for Stem Cell Research (IC-SCR) at Manipal Institute of Regenerative Medicine, MAHE – Bengaluru. See the Table of Materials for details about all materials and reagents used in this protocol.
1. Preparation of reagents and buffers
TYPE OF MEDIA | PURPOSE OF MEDIA | COMPOSITION FOR 50 mL | ||||||||
FBS | Pen-Strep | L-Glutamine | BASAL MEDIUM FOR UNDIFFERENTIATED hESCs | |||||||
10% media | MSC culture and maintenance | 5 mL | 500 μL | 500 μL | 44 mL | |||||
20% media | CFU-F assay | 10 mL | 500 μL | 500 μL | 39 mL | |||||
Serum starved (2%) media | Media for Control wells in Differentiation protocol | 1 mL | 500 μL | 500 μL | 48 mL | |||||
Neutralizing media | Media for neutralizing the cell suspension after trypsinization | – | 500 μL | 500 μL | 49 mL |
Table 2: Cell culture media for culture maintenance and assays. Abbreviations: MSC = mesenchymal stem cell; CFU-F = colony-forming unit-fibroblast.
COMPONENTS | OSTEOGENIC MEDIA | CHONDROGENIC MEDIA | ADIPOGENIC MEDIA |
Basal Media | 90 mL | 90 mL | 90 mL |
Induction media | 10 mL | 10 mL | 10 mL |
Total Volume | 100 mL | 100 mL | 100 mL |
Table 3: Differentiation media for trilineage differentiation of SHEDs.
2. Culture and maintenance of SHEDs
3. Characterization of MSCs
4. Cell surface staining for immunophenotyping
NOTE: Recommended cell culture plates for getting an optimal number of cells in steps 4.2-4.5 are 100 mm dishes or T75 flasks.
Tube type | Positive Comp Beads* | Negative Comp Beads* | Cells | Antibody added | |
FITC tube | 1 drop | 1 drop | – | Anti-human CD90-FITC (2 µL) | |
V450 tube | 1 drop | 1 drop | – | Anti-human CD44-V450 (2 µL) | |
PerCP-Cy 5.5 tube | 1 drop | 1 drop | – | Anti-human CD73-PerCP Cy 5.5 (2 µL) | |
PE tube | 1 drop | 1 drop | – | Anti-human CD45-PE (2 µL) | |
APC tube | 1 drop | 1 drop | – | Anti-human CD105-APC (2 µL) | |
DAPI tube | – | – | 50 µL | – | |
Unstained tube | – | – | 50 µL | – | |
*1 drop= 60 µL of bead suspension |
Table 4: Compensation control samples. Abbreviations: Comp = compensation; DAPI = 4',6-diamidino-2-phenylindole; FITC = fluorescein isothiocyanate; APC = allophycocyanin; PE = phycoerythrin; PerCP = peridinin-chlorophyll-protein.
TUBE TYPE | ANTIBODY AGAINST POSITIVE MARKER (2 µL) | ANTIBODY AGAINST NEGATIVE MARKER (2 µL) | ISOTYPE ANTIBODIES ADDED (2 µL) | TOTAL VOLUME OF ANTIBODIES | VOLUME OF CELL SUSPENSION ADDED | VOLUME OF STAINING BUFFER ADDED | ||
CD90-FITC FMO tube | – Anti-human CD44-V450 | Anti-human CD45-PE | FITC IgG1 isotype | 10 µL | 50 µL | 40 µL | ||
– Anti-human CD73 PerCP Cy 5.5 | ||||||||
– Anti-human CD105-APC | ||||||||
CD73-PerCP Cy5.5 FMO tube | – Anti-human CD44-V450 | Anti-human CD45-PE | PerCP Cy 5.5 IgG1 isotype | 10 µL | 50 µL | 40 µL | ||
– Anti-human CD90-FITC | ||||||||
– Anti-human CD105-APC | ||||||||
CD44-V450 FMO tube | – Anti-human CD90-FITC | Anti-human CD45-PE | V450 IgG1 isotype | 10 µL | 50 µL | 40 µL | ||
– Anti-human CD73-PerCP Cy 5.5 | ||||||||
– Anti-human CD105-APC | ||||||||
CD105-APC FMO tube | – Anti-human CD44-V450 | Anti-human CD45-PE | APC IgG1 isotype | 10 µL | 50 µL | 40 µL | ||
– Anti-human CD90-FITC | ||||||||
– Anti-human CD73-PerCP Cy 5.5 | ||||||||
CD45-PE FMO tube | – Anti-human CD44-V450 | – | PE IgG1 isotype | 10 µL | 50 µL | 40 µL | ||
– Anti-human CD90-FITC | ||||||||
– Anti-human CD73-PerCP Cy 5.5 | ||||||||
– Anti-human CD105-APC |
Table 5: FMO control samples. Abbreviations: FMO = fluorescence minus one; FITC = fluorescein isothiocyanate; APC = allophycocyanin; PE = phycoerythrin; PerCP = peridinin-chlorophyll-protein.
TUBE TYPE | ANTIBODY AGAINST POSITIVE MARKER (2 µL) | ANTIBODY AGAINST NEGATIVE MARKER (2 µL) | TOTAL VOLUME OF ANTIBODIES | VOLUME OF CELL SUSPENSION ADDED | VOLUME OF STAINING BUFFER ADDED | |
Mixed tube 1 | – Anti-human CD44-V450 | Anti-human CD45-PE | 10 µL | 50 µL | 40 µL | |
– Anti-human CD90-FITC | ||||||
– Anti-human CD73-PerCP Cy5.5 | ||||||
– Anti-human CD105-APC | ||||||
Mixed tube 2 | – Anti-human CD44-V450 | Anti-human CD45-PE | 10 µL | 50 µL | 40 µL | |
– Anti-human CD90-FITC | ||||||
– Anti-human CD73-PerCP Cy5.5 | ||||||
– Anti-human CD105-APC |
Table 6: Sample tubes for multicolor immunophenotyping of SHEDs. Abbreviations: SHEDs = stem cells from human exfoliated deciduous teeth; PE = phycoerythrin.
TUBE TYPE | ANTIBODY AGAINST POSITIVE MARKER (3 µL) | ANTIBODY AGAINST NEGATIVE MARKER (3 µL) | TOTAL VOLUME OF ANTIBODIES | VOLUME OF CELL SUSPENSION ADDED | VOLUME OF STAIN BUFFER ADDED | |
Mixed tube 1 | – Anti-human CD90-FITC | Anti-human CD45-PE | 9 µL | 50 µL | 41 µL | |
– Anti-human CD73-PerCP Cy5.5 | ||||||
Mixed tube 2 | – Anti-human CD90-FITC | Anti-human CD45-PE | 9 µL | 50 µL | 41 µL | |
– Anti-human CD73-PerCP Cy5.5 |
Table 7: Single-cell sorting reaction tubes. Abbreviations: FITC = fluorescein isothiocyanate; PE = phycoerythrin; PerCP = peridinin-chlorophyll-protein.
5. Single-cell sorting
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 properties of the SHEDs were determined from the CFU-F Assays shown in Figure 1C–E. As per the ISCT criteria, the multipotent MSCs were able to differentiate into the three mesodermal-derived lineages. Cytochemical staining with Oil Red O was used to detect the oil droplets in the differentiated adipocytes (Figure 1F); von Kossa staining was used to stain the calcium deposits found in the matrix of the differentiated osteoblasts (Figure 1G); and the aggrecans in the 3D culture were stained for Alcian Blue in the differentiated chondroblasts (Figure 1H).
To characterize the immunophenotype of the SHEDs, multiparametric flow cytometry assays were set. The experiments were performed along with the use of four types of experimental controls, namely compensation controls (Table 4), FMO and isotype controls (Table 5), and unstained controls. The isotypes of all the MSC and HSC antibodies used were added along with the FMO tubes to discriminate positive versus negative signals and high versus dim antigen expression. Figure 2A shows the distribution of the FMO and isotype controls used per experiment. The representative density plots showed no expression of the FITC isotype of CD90 FITC antibodies. Similarly, the histogram overlays (Figure 2B) compared the expression levels of the stained samples along with their controls in the respective channels of FITC, PerCP Cy5.5, and PE. Positive expression of the markers CD90 and CD73 (red histograms) and negative expression of CD45 are comparable with that of their unstained and FMO controls (blue and black histograms, respectively).
Flow cytometry-based characterization of the immunophenotypes has shown the marker distribution from one of the early passages (P6) SHEDs (Figure 3A–H). Both the ISCT-recommended markers and CD44 determined the parent populations as MSCs. The tetra-positive population demonstrated in the density plots showed ≥98% expression of CD90+CD73+CD105+CD44+ and ≤2% expression of CD45 markers. More than five independent experiments showed similar results. The late-passage SHEDs (P12), which showed differential expression of the positive markers, were chosen for single-cell sorting of high and dim expressors (Figure 4A–H). Following the gating strategy (Figure 4H), singlets→ Live cells→ Non-HSCs→ Double positive CD73+CD90+ and single positive CD73+CD90– populations were sorted using the single-cell sort mode. The sorted populations were collected in 96-well/48-well/6-well plate layouts with different seeding counts per well (Supplementary File 1). Three consecutive attempts were made in a single phase of sorting experiments and the survivability of cells post sort (in the double-positive expressors: CD90+CD73+) per experiment was 100%. Three independent phases of experiments have been conducted. We matched the number of cells that grew per well post sort to the number of cells sorted per well.
Figure 5A shows the CD90+CD73+ post-sorted cells collected in the 96-well plate showed adherence and proliferation like their parent population. The sorted cells have been observed to reach 70-80% confluency on the sixth day post sorting. The adhered and proliferating post-sorted cells were differentiated in the presence of adipogenic growth factors and then stained post Day 15 with Oil Red O using appropriate post-sorted control (undifferentiated) cells as well (Figure 5B,C).
Figure 1: Characterization of stem cells from human exfoliated deciduous teeth. (A) Immunofluorescence staining confirms that the isolated MSCs express vimentin (red, type III intermediate filaments), actin filaments (Alexa fluor 488 Phalloidin Probes), and DAPI (blue, nuclei). Photomicrograph obtained at original magnification of 20x. Scale bar = 10 µm. (B) Short-term cell growth assay of SHEDs exhibits their high proliferative capacity, as observed by the significant increase in the number of cells in culture by day 2 and a 14.3-fold increase in proliferation rate at the end of day 8 (n = 3, p-value<0.01). Standard deviation error bars have been shown. (C–E) CFU-F assay and Crystal violet staining confirm the colony-forming ability of SHEDs; (C) multiple colonies are expected to form after 14 days in culture; (D) single clonogenic colony originating from a single cell; (E) Crystal violet-stained single colony of SHEDs displays the typical fibroblast-like morphology of the cells. (F–H) MSCs under appropriate in vitro conditions are expected to undergo differentiation into adipogenic, osteogenic, and chondrogenic lineages by day 21. Cytochemical staining shows (F) Oil Red O (inset shows 40x image of adipocyte showing oil droplets), (G) von Kossa, and (H) Alcian Blue staining for adipogenic, osteogenic, and chondrogenic lineages, respectively, at Day 22. Photomicrographs are obtained at an original magnification of 20x. Scale bar = 10 µm. Abbreviations: MSCs = mesenchymal stem cells; DAPI = 4',6-diamidino-2-phenylindole; SHEDs = stem cells from human exfoliated deciduous teeth; CFU-F = colony-forming unit-fibroblast. Please click here to view a larger version of this figure.
Figure 2: Negative controls used in multiparametric experiment. (A) Fluorescence Minus One controls have been displayed in a tabular form, where FMO controls have been replaced by their specific isotypes. The representative density plots (left to right) show the unstained control (left), FMO of CD90 FITC (middle) with all antibodies added except CD90 FITC along with the addition of CD90 FITC isotype, and fully stained sample with all antibodies of the panel added (right). (B) Histogram overlay represents the comparison of three types of samples, black represents FMO, blue represents unstained, and red represents fully stained samples in their respective channels. Abbreviations: FMO = Fluorescence Minus One; FITC = fluorescein isothiocyanate; PerCP = peridinin-chlorophyll-protein; PE = phycoerythrin. Please click here to view a larger version of this figure.
Figure 3: Multiparametric immunophenotyping of SHEDs (P6). (A) SSC-A versus FSC-A: Based on their side and forward scatter properties, cells of interest are gated and separated from the debris; (B) FSC-H versus FSC-A and (C) SSC-H versus SSC-A:The two plots are employed for doublet discrimination, which enables the selection of singlets while eliminating aggregates that may generate false positive signals; (D) SSC-A versus CD45 PE: Exclusion gating allows selection of the CD45– population from the singlets; (E) CD73 PerCP-Cy5.5 versus CD90 FITC: From the CD45– live singlets, CD90+CD73+ cells are gated; (F) CD44 V450 versus CD105 APC: The CD90+CD73+ population, is further gated to define the tetra-positive CD90+CD73+CD44+CD105+ cells; (G) SSC-A versus Time(s): The plot depicts the stable acquisition over time (seconds) and that no events caused pressure fluctuations while acquisition; (H) Gating hierarchy. Abbreviations: SHEDs = stem cells from human exfoliated deciduous teeth; FITC = fluorescein isothiocyanate; APC = allophycocyanin; PE = phycoerythrin; PerCP = peridinin-chlorophyll-protein; SSC-A = side scatter-peak area; FSC-A = forward scatter-peak area; FSC-H = forward scatter-peak height. Please click here to view a larger version of this figure.
Figure 4: Single-cell sorting of immunophenotyped SHEDs (P12). (A–F) A step-by-step exclusion-based gating strategy to sort pure MSCs in the following sequential order: Cells to singlets_1, to singlets_2, to DAPI– live cells, to CD45– non-Hematopoietic cells, and finally to CD90+CD73+ cells. The gated populations shown in (F) were sorted using single-cell sort mode. (G) The SSC-A versus Time(s) plot describes the uninterrupted sorting of the samples. (H) Gating hierarchy. (n=3, Sort efficiency = 100%). Abbreviations: SHEDs = stem cells from human exfoliated deciduous teeth; MSCs = mesenchymal stem cells; DAPI = 4',6-diamidino-2-phenylindole; FITC = fluorescein isothiocyanate; APC = allophycocyanin; PE = phycoerythrin; PerCP = peridinin-chlorophyll-protein; SSC-A = side scatter-peak area. Please click here to view a larger version of this figure.
Figure 5: Morphological and functional characterization of post-sorted SHEDs. (A) Phase contrast images of post-sorted populations were collected in a 96-well plate on Day 6 at an original magnification of 10x, Scale bar = 10 µm (inset shows a 20x image depicting the sorted single cells changing morphology into fibroblast-like MSCs). Cytochemical staining for adipogenic differentiation using Oil Red O observed in (B) undifferentiated (control) and (C) differentiated post-sort population at an original magnification of 20x, Scale bar = 10 µm (inset shows a 40x image depicting the oil droplets observed). Abbreviations: SHEDs = stem cells from human exfoliated deciduous teeth. Please click here to view a larger version of this figure.
Supplementary File 1: Compiled sort reports generated per experiment. Please click here to download this File.
Supplemental Table S1: Optical Configuration of BD FACSAria Fusion. Abbreviations: LP = long pass; PMT = photomultiplier tube; DAPI = 4',6-diamidino-2-phenylindole; FITC = fluorescein isothiocyanate; APC = allophycocyanin; PE = phycoerythrin; PerCP = peridinin-chlorophyll-protein. Please click here to download this File.
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 this manuscript helps characterize SHEDs using a multiparametric approach and further sort populations of interest using the single-cell sort approach. However, the application of this protocol is not only limited to oral MSCs but also can be used for immunophenotyping and sorting of MSCs from other source tissues in the human body23.
Several critical steps in this protocol need to be discussed here for its successful implementation for the biological problem addressed. The key to every sorting protocol is to check two important components: its biological controls and the experimental controls. To begin with, clean culture maintenance ensures that the sample used does not get compromised while being sorted. The cell viability in the single-cell suspension pre-sorting shall ensure that more than 70% viable cell populations are started with, followed by analysis and sorting of healthy subpopulations. The stain buffer used in the single-cell suspension should contain 2% FBS and experiments are best done at room temperature. Selection of the correct confluence of these primary cells (80-85% confluency) avoids compromising their cell viability and reduces the chances of large aggregates. Additionally, the use of a live/dead marker (DAPI in this protocol) is essential for every sorting experiment. In this study, as the samples used were MSCs with an approximate diameter of 20-35 μm, the nozzle size chosen was 100 μm to sort them at a pressure of 20 psi on the BD FACSAria Fusion. This allowed cell sorting without compromising the cell viability during the process at lower sheath pressure. For detailed sort setup conditions, it is recommended to refer to the software BD FACSDiva guide24.
An essential prerequisite for multicolor experiments in flow cytometry is the need for appropriate controls; there are four major types of controls used here: autofluorescence, isotypes, FMO, and compensation controls. The auto-compensation matrix allowed accurate compensation, preventing spectral bleedthrough in the detectors, and used both compensation beads and cells for this purpose. The unstained sample set the threshold for autofluorescence of the MSCs. The mixed tubes with all four antibodies include the isotype and FMOs of each antibody used. DAPI compensation control was performed using MSCs, which were processed separately (with heat shock). The antibody panel (as indicated in Table 8) used here is specifically designed for hMSCs based on the criteria stated by the ISCT and the instrument configuration of the BD FACSAria Fusion cell sorter (refer to Supplemental Table S1). This is very crucial during the planning of multiparametric flow cytometry experiments. The characterization panel used for immunophenotyping consists of four positive MSC markers (CD90, CD105, CD73, and CD44) and one negative marker (CD45). In contrast, the panel for the sorting experiment contains two positive markers (CD90 and CD73), one negative marker (CD45), and a live/dead marker (DAPI) to ensure a high count of live MSCs.
Antibody/Marker | Fluorochrome | hMSC Surface Profile | Laser Wavelength (nm) | Excitation peak (nm) | Emission peak (nm) |
CD44 | V450 | Should express; >95% | 405 | 405 | 450 |
CD90 | FITC | 488 | 495 | 519 | |
CD73 | PerCP-Cy5.5 | 488 | 488 | 695 | |
CD105 | APC | 640 | 650 | 660 | |
CD45 | PE | Should NOT express; <2% | 561 | 566 | 574 |
DAPI | – | Dead cell marker | 405 | 358 | 461 |
Table 8: Multicolor panel designed for characterization and sorting of SHEDs by flow cytometry. Abbreviations: FITC = fluorescein isothiocyanate; APC = allophycocyanin; PE = phycoerythrin; PerCP = peridinin-chlorophyll-protein.
Selection of the sort purity mask
Considering the unpredictability of cellular heterogeneity in MSCs, bulk sorting may not necessarily achieve the level of purity among the sorted populations. We optimized the single-cell sorting protocol in this study to identify the single-cell clones and track their functional properties post sort in terms of potency. Single-cell sorting was performed on a BD FACSAria Fusion, which had the flexibility of the following settings: choice of 100 µm nozzle, 20 psi sheath pressure, a flow rate that is adjustable (maintained at 20 μL/min), and threshold rate at 380 events/s. The chances of selecting two target cells instead of one are minimal in this method. Normally, the single-cell sort mode of the algorithm is selected that can resolve the sorting of pure populations of target cells and consider how many drops will be sorted into the collection device of choice. The Precision mode ultimately should be accurately chosen such that it can combine the masks available in the sorting algorithms and create a stringent sorting condition to sort the purest populations of interest. Single-cell sort mode does not compromise on a target cell if it is aggregated to a non-target cell while sorting and that achieves the level of purity we look forward to in this approach. This was the module selected in this protocol and the sorted cells were collected in 96-well, 48-well, 24-well, and 6-well plate devices.
In this study, we sorted subpopulations based on their differential immunophenotype, namely CD90+CD73+CD45–. In both characterization and sorting protocols, our gating strategy (as shown in Figure 3 and Figure 4) has been based on exclusion gating from the CD45– population to give us the non-hematopoietic population (as these have been validated and characterized prior with the ISCT prescribed markers), after the identification of singlets. Eventually, the MSCs were identified from the CD45– population using the positive ISCT markers. It is worth noting here that there is not much difference in percentages in first gating the positive expressors of ISCT markers followed by the negative expressors of CD45. However, as the purpose of this sorting has been to identify cells that show altered expression of the known MSC markers, we have concluded our analysis with the positive expression plots.
In the representative figures 97% of the CD45– cells are MSCs, but during quantification of the antigen expressions, we have seen 75% of the cells are double-positive expressors of the two markers CD90 and CD73, while there are several populations that are single-positive expressors of the same markers (shown in Figure 3F) and few cells which do not express the markers at all.
During the characterization of these cells, we have identified the subpopulations as tetra-positive MSCs, triple-positive and double-negative populations. This differential expression of MSC markers expands with increasing passages and thus the need comes to study and correlate this difference with their functional properties. The ultimate purpose of sorting MSCs in our study is to create a comparative analysis between the identified subpopulations and determine whether the differential expressions of positive markers have functional implications.
The authors have nothing to disclose.
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.
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 |