Isolation and Validation of Podoplanin-positive Mesenchymal Stem Cell Subpopulations Using an Indirect Magnetic Bead Strategy

Summary

This protocol describes a magnetic bead-based sorting method using phycoerythrin (PE)-labeled flow cytometry antibodies and anti-PE microbeads to isolate specific mesenchymal stem cell subpopulations with high postsort target-marker positivity and preserved CCK-8-based proliferation/metabolic activity under the tested conditions, without requiring expensive cell sorters or target-specific custom beads.

Abstract

The study of functional heterogeneity in mesenchymal stem cells relies on the efficient isolation of specific subpopulations (e.g., podoplanin (PDPN)-positive cells). However, for most membrane protein targets, directly available commercial magnetic beads for sorting are limited. To address this, our laboratory has established and tested a standardized magnetic bead sorting protocol based on the indirect coupling principle of "phycoerythrin (PE)-labeled flow cytometry antibodies/anti-PE magnetic beads." Using PDPN-positive cells as a model target population, this protocol enriches a target cell fraction without relying on expensive flow cytometric cell sorters.

Researchers can use compatible PE-labeled flow cytometry antibodies targeting accessible cell-surface proteins and combine them with commercially available anti-PE microbeads to establish a sorting workflow for selected targets after target-specific optimization. This approach avoids the lengthy lead times and high costs associated with customizing or ordering target-specific magnetic beads for each new target, making it particularly suitable for accessible surface markers that are rare or understudied. In the PDPN model system tested here, the protocol enabled enrichment of the target subpopulation with high post-sort PDPN positivity and preserved CCK-8-based proliferation/metabolic activity compared with unsorted MSCs.

Introduction

In the field of orthopedics, injuries such as cartilage damage and lesions in the avascular zone (white zone) of the meniscus often result in irreversible joint destruction and loss of function, ultimately leading to osteoarthritis. This is largely attributable to the poor regenerative capacity of these tissues, which stems from a lack of direct blood and lymphatic supply, as well as the presence of highly differentiated cells with limited capacity for proliferation and migration^1,2,3,4. To address this challenge, various clinical strategies have been explored, including the use of exosomes^5,6, autologous or allogeneic chondrocyte transplantation^7,8, and tissue engineering approaches^9,10,11,12. In recent years, mesenchymal stem cells (MSCs) have emerged as a promising cell source for regenerative repair, owing to their multilineage differentiation potential and immunomodulatory properties, offering new hope for cartilage regeneration^13,14,15,16.

Despite their therapeutic potential, MSCs exhibit significant heterogeneity. Even within a single tissue source, such as bone marrow or umbilical cord, distinct subpopulations of MSCs can display considerable variability in proliferation capacity, multilineage differentiation potential (particularly chondrogenic differentiation), and in vivo regenerative function^17,18,19. Therefore, the precise identification and isolation of functionally defined MSC subpopulations—such as those with enhanced chondrogenic capacity—is essential for advancing our understanding of their biological properties and improving the efficacy of cartilage repair. This represents a critical step in our research.

As research progresses, an increasing number of novel membrane proteins have been identified as markers of functionally distinct MSC subpopulations. However, translating these discoveries into effective cell sorting strategies remains technically challenging. Conventional fluorescence-activated cell sorting (FACS), while powerful, requires specialized instrumentation and technical expertise, limiting its accessibility in many laboratories. On the other hand, immunomagnetic cell sorting—though more widely used—is constrained by the limited availability of commercial reagents, which are primarily designed for a few well-established membrane targets. Consequently, for many newly discovered markers with potential biological significance, there are no commercially available magnetic beads, making it difficult to specifically enrich these valuable MSC subpopulations.

To overcome this limitation, the present study aims to establish and standardize a flexible, cost-effective, and efficient protocol for positive cell selection. This approach is based on the indirect conjugation of commercially available phycoerythrin (PE)-conjugated antibodies with anti-PE immunomagnetic beads. Previous reports have demonstrated that podoplanin-positive (PDPN⁺) MSCs possess superior stemness maintenance and differentiation capacity compared to their PDPN^- counterparts²⁰. Furthermore, the involvement of PDPN⁺ MSCs in cartilage regeneration and the progression of osteoarthritis (OA) has been documented in the literature^21,22. Using podoplanin (PDPN)—a recently identified candidate marker of functional MSC subpopulations—as a model target, we sorted PDPN-positive cells from bone marrow-derived MSCs. We provide a detailed step-by-step protocol covering single-cell suspension preparation, antibody and magnetic bead labeling, and magnetic separation. Moreover, we evaluate the yield, post-sort PDPN positivity, and CCK-8-based proliferation/metabolic activity of the sorted cells. By standardizing this protocol, we aim to provide researchers with a versatile, accessible method for isolating target cell subpopulations using compatible antibodies against accessible cell-surface markers, thereby facilitating fundamental stem cell research and future translational applications in regenerative medicine.

Protocol

Human bone marrow-derived MSCs were commercially obtained (see the Table of Materials). The supplier confirmed MSC identity by positive expression of CD73, CD90, and CD105; negative expression of CD34, CD45, and HLA-DR; and trilineage differentiation potential. Cells were expanded in culture and used at passages 3–5. All human-derived cell work was performed in a Class II biological safety cabinet under sterile conditions. As the cells were commercially acquired and no human subjects were directly recruited or sampled in this protocol, no direct IRB approval was required.

1. Preparation of single-cell suspension

NOTE: The quality of the single-cell suspension directly affects the sorting efficiency. Ensure cells are well-dispersed, highly viable, and free of clumps. Visible clumps should be dispersed by repeated gentle pipetting until clumps are no longer visible.

1. Select MSCs (passages 3–5) that have reached 80–90% confluence. Discard the culture medium.
2. Wash the cells with 2 × 10 mL of prewarmed PBS. Gently rock the vessel and discard the PBS.
3. Add 2 mL of 0.25% Trypsin-EDTA and incubate at 37 °C for 3–5 min. Observe the cells under a microscope until they round up and begin to detach.
4. Add 4 mL of culture medium containing 16.5% FBS (see the Table of Materials) to neutralize the trypsin. Gently pipette the cells to ensure complete detachment. Transfer the cell suspension to a 15 mL centrifuge tube.
   NOTE: Check under the microscope to confirm that all cells have been collected.
5. Centrifuge at 300 × g for 3 min and discard the supernatant.
6. Resuspend the cell pellet in 1 mL of Buffer (see the Table of Materials). Proceed to cell counting.

2. Cell counting and concentration adjustment

NOTE: If the total cell number is less than 1 × 10⁷, still add the antibody and magnetic reagents based on the standard volume for 1 × 10⁷ cells to ensure labeling efficiency.

1. Centrifuge the cell suspension again at 300 × g for 3 min and discard the supernatant.
2. Resuspend the cells in Buffer to achieve a density of 1 × 10⁸ cells/mL.
   NOTE: For example, to process 1 × 10⁷ cells, resuspend the pellet in 100 µL of buffer.
3. Transfer the cell suspension to a 5 mL polystyrene round-bottom tube.

3. Antibody labeling

1. Add 10 µL of Fc gamma RII Blocker (see the Table of Materials) per 1 × 10⁷ cells. Mix gently and incubate at room temperature for 10 min.
2. Add 5 µL of PE-conjugated anti-human PDPN antibody (see the Table of Materials) per 1 × 10⁷ cells, following the manufacturer's recommended concentration. Mix gently and incubate at room temperature for 15–20 min, protected from light.
   NOTE: The antibody volume should be adjusted according to the datasheet of the specific flow cytometry antibody being used, as concentrations may vary between products.

4. Magnetic bead labeling

NOTE: Keep all reagents on ice before use and return them to ice immediately after each addition to maintain activity and consistency.

1. Add 10 µL of Selection Cocktail per 1 × 10⁷ cells (see the Table of Materials). Mix gently and incubate at room temperature for 15 min.
2. Add 5 µL of magnetic beads per 1 × 10⁷ cells (see the Table of Materials). Mix gently and incubate at room temperature for 10 min.
   NOTE: Vortex the magnetic beads vial vigorously for 30 s before use to ensure even dispersion.

5. Magnetic separation

1. Place a 5 mL polystyrene round-bottom tube containing the labeled cell sample in the magnetic separation stand (see the Table of Materials).
2. Add buffer to the tube to bring the total volume to 2.5–3 mL. Incubate for 5 min at room temperature.
3. Perform the separation: Holding the magnet and tube together as one unit, invert them to pour the supernatant into a new collection tube. This collected fraction contains the PDPN-negative cells; keep this tube on ice.
   NOTE: Do not remove the tube from the magnetic stand during this step, as this will disrupt the separation and cause the procedure to fail.
4. Repeat steps 5.1–5.3 3x. Each time, add buffer to the original tube in the magnet, wait 5 min, and pour the supernatant into the same negative fraction collection tube used in step 5.4.
5. After the final wash, remove the tube containing the magnetically bound cells from the stand. This tube contains the PDPN-positive cell fraction.
6. Add 3 mL of buffer to resuspend the positive cells. Transfer this suspension to a fresh, labeled tube (the positive fraction tube).

6. Collection, culture, and validation of target cells

1. Count the cells from both the positive and negative fraction tubes. Record the cell yield for each fraction.
2. Centrifuge the positive fraction cell suspension at 300 × g for 3 min. Discard the supernatant.
3. Resuspend the cell pellet in MEM Alpha culture medium containing 16.5% FBS. Adjust the cell density as needed and seed the cells into culture plates or flasks.
4. Incubate the cells at 37 °C in a 5% CO₂ incubator.

7. Sample preparation for purity and functional validation

1. Count the cells and seed them onto a 100 mm culture dish at a density of 3 × 10⁶ cells per dish in 12 mL of culture medium. Twenty-four hours after seeding, observe cell attachment. Replace the culture medium with fresh medium to remove any residual magnetic beads and non-adherent cells.
2. Harvest the cells by trypsinization. Gently pipette up and down 10–15x to dislodge and remove magnetic beads and residual antibodies.
3. Assess the percentage of PDPN-PE-positive cells by flow cytometry (see Supplemental File 1 for the detailed protocol).
4. Collect cells during the logarithmic growth phase. Seed them into a 96-well plate. At different time points (0 h, 24 h, 48 h, 72 h), add CCK-8 reagent (see the Table of Materials) and measure the absorbance according to the manufacturer's instructions. Plot a proliferation curve based on the data.

Results

The overall principle and workflow of the experiment are illustrated in Figure 1. All subsequent experimental data are based on passage 5 cells. Following magnetic separation, the obtained PDPN⁺ cells were cultured for 24–48 h. After replacing the culture medium to remove non-adherent cells and potential residual magnetic beads (Figure 2A), we evaluated the sorting efficiency and cell yield. Starting with an initial population of 1 × 10⁷ MSCs, the final cell count after Fc receptor blocking, antibody labeling, and magnetic separation was 3.5 ± 0.21 × 10⁶ for the PDPN⁺ fraction and 6.4 ± 0.15 × 10⁶ for the PDPN^- fraction (Figure 2B).

Before magnetic sorting, the frequency of PDPN-positive cells in the starting cell population was determined by flow cytometry to be 38.7% (Supplemental File 1—Supplemental Figure S1A). After sorting, the positive fraction contained 97.9% PDPN-PE-positive cells (Figure 2C), corresponding to an enrichment fold of 2.5-fold relative to the pre-sort frequency. Based on the starting PDPN-positive frequency and the number of cells recovered in the positive fraction, the estimated recovery of the PDPN-positive fraction was approximately 90%. Flow cytometry analysis of the negative fraction revealed that 4.30% of PDPN⁺ cells remained, indicating efficient depletion of PDPN+ cells from the negative fraction (Supplemental File 1—Supplemental Figure S1B). These data collectively demonstrate efficient enrichment of PDPN⁺ cells and depletion of PDPN⁺ cells from the negative fraction.

We further investigated whether the immunomagnetic separation process affects cellular function by assessing the proliferative capacity of sorted PDPN⁺ cells using the CCK-8 assay. Pre-sort and post-sort cells were seeded at the same density and cultured under identical conditions before the CCK-8 assay. As shown in Figure 2D, the proliferation curve of PDPN⁺ cells over three consecutive days of culture showed no significant difference compared to unsorted MSCs (P > 0.05). These findings indicate that under the tested conditions, the sorting strategy did not cause a detectable reduction in CCK-8-based proliferation/metabolic activity compared to unsorted MSCs.

A successful sorting experiment typically yields a post-sort PDPN+ percentage above 95% (as shown in Figure 2C). Suboptimal results may indicate specific technical issues. Low postsort PDPN⁺ percentage (<80%) often results from insufficient antibody labeling, incorrect antibody concentration, or inadequate washing steps. Low yield (<2 × 10⁶ PDPN⁺ cells from 1 × 10⁷ starting cells) may be caused by cell clumping before separation, excessive washing that dislodges magnetically bound cells, or incomplete trypsinization leading to cell loss. High nonspecific carryover (PDPN^- cells appearing in the positive fraction) typically indicates incomplete separation, often due to disturbing the tube during magnetic separation or using an incorrect buffer.

Figure 1. Experimental principle and flow chart. (A) One end of the anti-PDPN antibody binds to PDPN on the cell membrane, while the other end is conjugated to PE. Magnetic beads capture the target protein-expressing cells by binding to PE. (B) Overall workflow of magnetic bead screening. Abbreviations: PDPN = podoplanin; PE = phycoerythrin. Please click here to view a larger version of this figure.

Figure 2. Magnetic bead screening result graph. (A) Magnetic beads before and after medium change. Cells before (left) and after (right) medium change. Beads are visible on the left and absent on the right. Scale bar = 50 µm. (Magnification: 40×) (B) Bar graph showing the number of PDPN⁺ and PDPN^- cells obtained after magnetic-activated cell sorting. (C) Flow cytometric analysis of PDPN-PE⁺ cells after sorting. (D) Cell proliferation assay. Line graph depicting the OD values (450 nm) measured at 0, 24, 48, and 72 h, showing no significant difference (ns) in CCK-8-based proliferation/metabolic activity between pre-sort and post-sort cells. Data are presented as mean ± SD from three independent replicates (n = 3). Statistical significance was determined by unpaired two-tailed Student's t-test. ns indicates not significant (p > 0.05). Abbreviations: PDPN⁺ = PDPN-positive; PDPN^- = PDPN-negative. Please click here to view a larger version of this figure.

Supplemental File 1: Flow cytometry validation and analysis of PDPN expression in human bone marrow mesenchymal stem cells. Please click here to download this file.

Discussion

In this study, we established a magnetic bead-based protocol for isolating PDPN+ cells from bone marrow-derived MSCs. This approach enables efficient enrichment of the PDPN-positive subpopulation while maintaining CCK-8-based proliferation/metabolic activity under the tested conditions.

Compared with conventional direct immunomagnetic sorting methods, the most significant advantage of this strategy lies in its versatility. Traditional approaches require the development or customization of specific magnetic beads for each new marker of interest²³, a process that is both time-consuming and costly. In contrast, this method leverages commercially available PE-conjugated antibodies—a common reagent in most laboratories—allowing researchers to establish a sorting workflow for selected surface targets after target-specific optimization. This flexibility is expected to render the protocol applicable not only to PDPN but also to other surface markers of MSCs (e.g., CD146, CD90, CD105)²⁴ or to subpopulation isolation from other cell types. Furthermore, compared with fluorescence-activated cell sorting (FACS), this method requires minimal specialized equipment and is technically straightforward, making it easily implementable in standard cell culture laboratories. This is particularly advantageous for research settings with limited access to advanced instrumentation.

This protocol is best suited for isolating viable cells expressing accessible surface antigens for which PE-conjugated antibodies are commercially available. It is also appropriate for routine enrichment of subpopulations where cell recovery, postsort culture, and moderate throughput are sufficient. However, this method is less suitable for the following situations. First, this method is dependent on PE-conjugated antibodies. If a target of interest lacks a commercially available PE-conjugated format, or if only antibodies conjugated to other fluorochromes (e.g., FITC, APC) are available, additional steps such as secondary antibody labeling or the purchase of corresponding secondary antibody-conjugated magnetic beads would be required, potentially increasing both procedural complexity and cost. Second, this protocol is specifically designed for cell-surface antigens; alternative strategies remain necessary for isolating cells based on intracellular markers. Third, while the yield of PDPN+ cells in this study was consistent with expectations, sorting efficiency may be influenced by several experimental variables, including antibody concentration, incubation time, and the thoroughness of washing steps. Therefore, when establishing a sorting protocol for a new target, we recommend performing preliminary optimization experiments to determine the ideal parameters. We acknowledge that residual magnetic beads could theoretically affect downstream functional assays. However, the standard washing and pipetting steps performed after sorting effectively removed most free or loosely bound beads, minimizing any potential impact on the proliferation results presented here. Additionally, post-sort viability was not directly measured; therefore, viability claims should be limited to the observation that sorted cells retained CCK-8-based proliferation/metabolic activity under the tested conditions.

Several critical steps influence sorting efficiency and reproducibility. First, cell dissociation quality is paramount; over-trypsinization reduces surface antigen integrity, while under-dissociation leaves clumps that cause column clogging. If clumps are visible, gentle pipetting (trituration) or filtration through a sterile cell strainer is recommended. Second, antibody titration is recommended for each new batch of PE-conjugated antibody to determine optimal concentration, incubation time, and washing stringency, using post-sort yield, purity, and nonspecific binding as readouts. Third, magnetic beads must be fully resuspended before use by vortexing or pipetting, as settling leads to inconsistent labeling. During the magnetic bead labeling step (protocol step 4), cells can be handled at room temperature. However, all reagents should be kept on ice before use and returned to ice immediately after each addition to maintain activity and consistency. Finally, avoid disturbing the column or tube during magnetic separation, as agitation can dislodge retained cells and reduce yield.

In conclusion, this study establishes and validates an indirect immunomagnetic sorting method based on PE-conjugated antibodies and anti-PE magnetic beads. This protocol enables efficient, high-purity enrichment of the PDPN+ subpopulation from MSCs without a detectable reduction in CCK-8-based proliferation/metabolic activity under the tested conditions. By combining the flexibility of antibody-based labeling with the gentle nature of magnetic separation, this approach provides a practical foundation for future investigations into the functional roles of PDPN+ MSCs in chondrogenesis, immunomodulation, and tissue repair. Subsequent studies will focus on evaluating the in vivo performance of PDPN+ cells in animal models and performing transcriptomic comparisons between PDPN+ and PDPN- subpopulations, with the goal of elucidating their distinct contributions to MSC heterogeneity and regenerative function.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
7-AAD Cell Viability Assay Kit	KeyGEN	KGD1202-50
Anti-human CD32 Blocker (Fc gamma RII Blocker)	STEMCELL Technologies	18520
Buffer	STEMCELL Technologies	20104	Buffer
CCK-8	Meilunbio	MA0218-5
Dextran Magnetic Particles	STEMCELL Technologies	50100	Magnetic Particles
Fetal Bovine Serum	Gibco	16050-122
Magnet	STEMCELL Technologies	18000	magnetic separation stand
MEM Alpha Medium	Gibco	C12571500BT	MEM Alpha Medium (82.5%) + FBS (16.5%) + Penicillin-Streptomycin (1.0%)
MSCs	KeyGEN	KGG5575-1	Human bone marrow-derived mesenchymal stem cells
PE-conjugated Anti-Human PDPN Antibody	ABclonal	A26770	PDPN-PE
PE Selection Cocktail	STEMCELL Technologies	18151	Selection Cocktail
Penicillin-Streptomycin	Gibco	15140122
Trypsin-EDTA	Gibco	25200-072