lineage-specific cytochemical staining of mesenchymal stem cells for characterizing differentiated cells

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

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.

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

This protocol details a crucial method for accurately characterizing and purifying human mesenchymal stem cells on a single platform, enabling efficient downstream applications like stem cell transplantation. It is particularly significant for clinical labs enhancing molecular and cytogenetic assays through precise single cell sorting of engineered or natural stem cells. The commonly used methods of isolation of purified stem cell populations refer to techniques such as immunomagnetic separation, density gradient, or microfluidic cell sorting.A technique like index sorting allows it to be linked to single cell RNA profiling that can extract transcriptomic information of each sorted cell. The key challenges involve maintaining sample quality, optimization of cell culture conditions, including cell density and viability during and after sorting is required. Additionally, choosing suitable nozzle sizes for the instrument and ensuring timely access to the sorters would also be considered.This protocol illustrates a precise staining panel for immunophenotyping and sorting the desired cell population. It ensures standardized sample quality, optimal cell viability, density for cell population sorting and define sorting experimental parameters on the FACSDiva software. This method enables a straightforward selection and immunophenotyping of stem cell population expressing specific biomarkers.This simple platform allows efficient sorting based on the target population aiding and obtaining purified samples for subsequent downstream applications.

Watch this Scientific Journal Video about Lineage-Specific Cytochemical Staining of Mesenchymal Stem Cells for Characterizing Differentiated Cells at JoVE.com

Lineage-Specific Cytochemical Staining of Mesenchymal Stem Cells for Characterizing Differentiated Cells  

To stain the 2D adipocyte lineage, add 200 microliters of permeable solution from kit to the plate. Next, incubate the cells in 200 microliters of Oil Red O Working solution for 10 minutes. Subsequently, wash the cells five times with 200 microliters of distilled water after removing the stain.To stain the 2D osteoblast lineage, add 200 microliters of 5%fresh silver nitrate to each well, then place the plate under ultraviolet light for an hour. Incubate the cells in 200 microliters of 2.5%sodium thiosulfate for five minutes before a distilled water rinse. For the 3D cultures, first, obtain the cryo sections of the pelleted cultures.To stain the cells of the chondrocyte lineage, add one to two milliliters of washing solution to the air dried sections. Next, incubate them in fixing solution for 30 minutes. After washing with distilled water, place the cells in one to two milliliters staining solution for 30 minutes.Rinse the cells three times with 0.1 normal hydrochloric acid before rinsing with distilled water. After 21 days, the MSCs under in vitro conditions differentiated into adipocytes, osteoblasts, and chondrogenic lineages.

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121 Views.  Manipal Academy of Higher Education, Manipal. To stain the 2D adipocyte lineage, add 200 microliters of permeable solution from kit to the plate. Next, incubate the cells in 200 microliters of Oil Red O Working solution for 10 minutes. Subsequently, wash the cells five times with 200 microliters of distilled water after removing the stain.To stain the 2D osteoblast lineage, add 200 microliters of 5%fresh silver nitrate to each well, then place the plate under ultraviolet light for an hour. Incubate the cells in 200 microliters of ...

Video: Lineage-Specific Cytochemical Staining of Mesenchymal Stem Cells for Characterizing Differentiated Cells  

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.

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 ...

A Step-by-Step Guide for Seeding and Differentiation of Mesenchymal Stem Cell Cultures for Characterization

To begin, seed mesenchymal stem cells into T-75 flasks, 100 mm dishes, and two 48-well plates for all the experiments. Add 200 microliters of 10%MSC culture media to the wells of the 48-well plates. When the cell monolayer reaches 90%confluency, replace the existing media of the designated control wells with 2%serum media.Add differentiation media of either adipogenic or osteogenic lineage into the last two wells labeled Test. On the same day, trypsinize a confluent 100 mm dish and transfer the cell suspension into two labeled 15-milliliter tubes. Centrifuge the tubes at 300 x g for six minutes.Then pipette 2 milliliters of the respective media into the control and test tubes. Place the loosely capped tubes in the incubator at 37 degrees Celsius under 5%carbon dioxide for 21 days with media replacement every three days. The SHEDs were characterized with standard immunofluorescence assays, showing the expression of vimentin, actin filaments, and nuclei stained with DAPI.Short-term cell growth assays showed an increased proliferation rate from day two to day eight. Colony-forming unit assay and crystal violet staining confirmed the colony-forming ability of SHEDs after 14 days.

Mesenchymal Stem Cell Surface Staining for Immunophenotyping

To begin, centrifuge the trypsinised mesenchymal stem cells at 300 g for six minutes to obtain the cell pellet. Next, resuspend the pellet in one milliliter of MSC media before cell counting. Centrifuge the cell suspension again, then resuspend the pellet in an appropriate volume of the staining buffer after discarding the supernatant.For the preparation of compensation controls, label seven fax tubes as unstained, DAPI, V450, FITC, PE, PerCP-Cy5.5, and APC. Next, prepare the single stain tubes for compensation and incubate them in the dark for 30 minutes. Following incubation, wash the cells in each tube two times and the beads one time with one milliliter of staining buffer.Next, centrifuge the vortexed tubes at 200 g for 10 minutes at room temperature. After discarding the supernatant, resuspend the pellet in 500 microliters of staining buffer. Incubate the DAPI tube in a 60 degree Celsius water bath for five minutes followed by a 15-minute incubation on ice to induce the inclusion of the dye.Add five microliters of DAPI to the suspension and incubate until acquisition. Prepare the cell and antibody suspension in five labeled fax tubes as per the table. Gently vortex the tubes before incubation.Then, wash each tube two times with one milliliter of the staining buffer. Centrifuge the vortex tube at 200 g for 10 minutes at room temperature, then resuspend the remaining pellet in 500 microliters of staining buffer.

Mesenchymal Stem Cell Sample Preparation, Flow Cytometric Analysis, and Cell Sorting

To begin, label two new fax tubes as mixed tubes one and two. Prepare the cell and antibody suspension as per the given table for flow cytometric analysis. After incubating the tubes for 30 minutes, wash each tube two times with one milliliter of staining buffer.Centrifuge the vortexed tubes at 200 G for 10 minutes at room temperature. Then re-suspend the pellet in 500 microliters of staining buffer. Coat the wells of a 48 well plate with 200 to 500 microliters of FBS, and keep for one hour.Then pipette 200 to 500 microliters of 10%MSC media after removing the residual FBS. To prepare the samples for single cell sorting, pipette one milliliter of FBS into two new fax tubes. Roll the tubes to create an even coat of the FBS on the tube's inner surface.Prepare the cell and antibody suspension in mixed tubes one and two, as per the table, for the sorting experiment. Then incubate the tubes in the dark for 30 minutes at room temperature. After washing with staining buffer, centrifuge the tubes at 200 G for 10 minutes at room temperature.Then re-suspend the pellet in 500 microliters of staining buffer and incubate for 15 minutes in five microliters of DAPI before sorting. To set up the compensation matrix, use the single stain compensation tubes. Click on experiment from the toolbar in the instrument software.Then click on compensation setup, followed by create compensation controls. Next, click on unstained tube, followed by parameters in the cytometer dialogue box. Adjust the voltages by editing the values for each parameter.Click on load in the acquisition dashboard and then click acquire. Drag the P-1 gate to the cell population and apply it across all compensation controls to set the voltage and negative gate for each parameter. Now load the single stain compensation tubes individually.Record the data and save it. Select the current experiment, right click on it, and select calculate compensation values, followed by link and save. Run the mixed tube one and record 10, 000 cells.Set the gates on the population of interest to be sorted with the proper gating strategy. Next, load a collection plate into the instrument and set a target cell number, ranging from 2, 500 to 5, 000 cells per well. Then select the single cell sort purity mask.Collect the sorted cell populations in a collection device, keeping them on ice for the duration of the sorting experiment. Finally, shift the plates into a 5%carbon dioxide incubator to maintain the cultures at 37 degrees Celsius. The multi-parametric flow cytometry assays plots showed no expression of the FITC isotype of CD 90 FITC antibodies.Positive expression of the markers CD-90 and CD-73 and negative expression of CD-45 are comparable with that of their unstained and FMO controls. The immuno phenotypes showed the marker distribution from one of the early passages with the ISCT recommended markers and CD-44, determining the parent populations as MSCs. The late passage sheds were used for single cell sorting of high and dim expressors.The post sorted cells collected in the 48 well plate showed adherence and proliferation, like their parent population. The post sorted cells differentiated in the presence of adipogenic growth factors.