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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119 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 ...