Transform MSC Research With Real-Time, Non-Invasive Differentiation Monitoring

Application Note Rapid Assessment of MSC Differentiation with Non-Invasive Monitoring and HTS Cytometry Authors: Natasha Lewis, Jasmine Trigg, Daryl Cole, Nicola Bevan Sartorius UK Ltd, Royston, Hertfordshire, UK Correspondence Email: askascientist@sartorius.com Abstract Mesenchymal stem cells (MSCs) are adult stem cells with multipotent differentiation potential and significant immunomodulatory capacity, making them highly valuable in regenerative medicine, tissue engineering, and cell therapy. Ensuring the therapeutic efficacy of MSCs necessitates rigorous confirmation of their differentiation potential into specific cell types, such as osteoblasts and adipocytes. Traditional endpoint assays for assessing MSC differentiation are limited in providing dynamic insights, highlighting the need for advanced technologies that offer real-time, high-throughput, and multiparametric analysis. This study utilizes the Incucyte® Live-Cell Analysis System and iQue® High-Throughput Screening Cytometer to monitor MSC differentiation in real-time. These technologies enable continuous live-cell imaging and rapid, multiplexed analysis, offering a comprehensive view of the differentiation process. The results demonstrate quicker confirmation of differentiation potential and preservation of cellular material for further analysis, enhancing the efficiency and reliability of MSC research. September 2024 Keywords or phrases: Mesenchymal stem cells, Differentiation, Cell therapy, Tissue engineering, Live-cell analysis, Osteogenesis, Adipogenesis For further information, visit sartorius.com 2 Introduction Mesenchymal stem cells (MSCs) are adult stem cells resident predominantly in the bone marrow alongside other sites including adipose tissue, umbilical cord blood, and dental pulp. They are self-renewing and have differentiation potential to the chondrocyte, osteocyte, and adipocyte lineages. MSCs have known capacities in immunomodulation and paracrine effects that stimulate tissue repair and regeneration. Their multi-potency and functionality make them of great interest in the fields of regenerative medicine, tissue engineering, and cell therapy, both in terms of direct repair and for treating inflammatory and autoimmune diseases. As the body of research into these unique cells grows, so does their medical potential. Clinical examples of the use of MSCs include treatment of osteoarthritis,¹ graft versus host disease,² and acute myocardial infarction.³ The therapeutic efficacy of MSCs is critically dependent on their differentiation potential into specific cell types, which is a highly regulated process influenced by various biochemical and physical cues. Osteogenic differentiation leads to the formation of osteoblasts, which are essential for bone formation and remodeling. Differentiation promoting factors elicit the expression of osteogenic markers including alkaline phosphatase (ALPL) and extracellular deposition of calcium phosphate (hydroxyapatite). Adipogenesis leads to the formation of adipocytes which play a vital role in tissue homeostasis by storing energy as fat. As such, key features of adipocytes include characteristic cytoplasmic lipid droplets, a rounded morphology, and expression of adipogenic markers and adipokines. The ability of MSCs to differentiate must be rigorously confirmed before they can be utilized in downstream applications, as it is a key characteristic used for their identification.⁴ Traditional methods for assessing MSC differentiation are often endpoint assays, which provide limited insights into the dynamic processes of differentiation. This limitation underscores the need for advanced technologies that can offer real-time, high-throughput, and multiparametric analysis. Addressing these challenges requires the development and adoption of standardized, reliable, and reproducible methods for MSC differentiation assessment, using advanced technologies such as the Incucyte® Live-Cell Analysis System (Incucyte® System) and iQue® HighThroughput Screening (HTS) Cytometer. Continuous livecell imaging allows researchers to monitor morphological changes and differentiation markers over time. This method provides valuable insights into the kinetics of cell differentiation, proliferation, and morphology, offering a dynamic view of cellular processes that static endpoint assays cannot capture. Meanwhile, HTS by cytometry facilitates rapid, multiplexed analysis of cellular phenotypes and secreted factors, providing a comprehensive view of the differentiation process. The iQue® platform’s highthroughput capabilities allow for the simultaneous analysis of multiple parameters across large sample sets, significantly enhancing the efficiency and depth of MSC research. Here, we present a comprehensive workflow to evaluate the functionality of MSCs. This workflow aims to provide robust and reliable methods for assessing MSC differentiation, thereby enhancing the reliability and efficiency of experimental workflows and ensuring the therapeutic potential of MSCs is fully realized. 3 Materials and Methods Media for Routine Cell Culture and for Differentiation Materials Supplier Cat. No. Final Concentration MSC NutriStem® XF Medium Sartorius 05-200-1A MSC NutriStem® XF Supplement Mix Sartorius 05-201-1U MSCgo™ Osteogenic Differentiation Medium Sartorius 05-440-1B MSCgo™ Rapid Osteogenic Differentiation Medium Sartorius 05-442-1B MSCgo™ Adipogenic SF, XF Supplement Mix I Sartorius 05-331-1-01 MSCgo™ Adipogenic SF, XF Supplement Mix II Sartorius 05-332-1-15 NutriCoat™ Attachment Solution Sartorius 05-760-1-15 1:500 Incucyte® Opti-Green Reagent (supplied with Incucyte® Mouse IgG1 Fabfluor Antibody Labeling Dye) Sartorius 4745 0.5 µM Calcein Merck CO875 1 µM BioTracker 488 Green Lipid Droplet Dye Merck SCT144 1:1,000 Bone marrow mesenchymal stem cells RoosterVial™-hBM RoosterBio MSC-003 Table 1: Cell culture media and reagents used for culturing MSCs. Surface Marker Expression Panel Reagents Marker Product Name Supplier Cat. No. Final Concentration Viability Zombie Violet™ Fixable Viability Kit BioLegend 423113 1 :200 MSC marker panel CD34 FITC anti-human CD34 Antibody Invitrogen MA5-16925 1:100 CD45 Brilliant Violet 570™ antihuman CD45 Antibody BioLegend 304033 1:100 CD73 APC anti-human CD73 (Ecto-5'-nucleotidase) Antibody BioLegend 344005 1:100 CD90 PE/Dazzle™ 594 anti-human CD90 (Thy1) Antibody BioLegend 328133 1:100 CD105 PE/Cyanine7 anti-human CD105 Antibody BioLegend 323217 1:100 Fixative Fixation buffer BioLegend 420801 1X Permeabilization buffer Intracellular Staining Perm Wash Buffer (10X) BioLegend 421002 1X Osteogenesis marker Alkaline phosphatase (ALPL) Human Alkaline Phosphatase/ALPL PE-conjugated Antibody R&D Systems FAB1448P-025 1:100 Adipogenesis marker Fatty Acid Binding Protein 4 (FABP4) Alexa Fluor® 647 Anti-FABP4 antibody Abcam ab216529 1:100 Table 2: Reagents used for cell analysis using the iQue® Cytometer. 4 MSC Culture Prior to seeding into culture vessels for routine culture and experiments, plates and flasks were pre-coated with NutriCoat™ Attachment Solution. The solution was diluted in PBS according to the recommended volumes, added to the culture vessel and incubated for 1 hour in a humidified CO₂ incubator (37°C). Prior to cell seeding, the solution was aspirated, and cell culture medium containing a cell suspension was quickly added to ensure that the coating did not dry out. MSCs were obtained from RoosterBio and cultured in MSC Nutristem® XF basal medium (basal medium). They were seeded at a density of 3,000 cells/cm² in tissue culture flasks, passaged at 80% confluency and analyzed using the Incucyte® System. MSC Differentiation MSCs were differentiated into osteoblasts using either MSCgo™ Osteogenic Differentiation Medium or MSCgo™ Rapid Osteogenic Differentiation Medium. Briefly, MSCs were seeded into 24-well plates pre-coated with MSC attachment solution at 6 × 10⁴ cells/well (3 × 10⁴/cm²) in basal medium. After 24 hours, or when the cells had reached 80% confluency, the medium was exchanged to differentiation medium containing Incucyte® Opti-Green (background suppressor) and calcein. Cells were cultured in differentiation medium for either 21 or 10 days, respectively. MSCs were differentiated into adipocytes using MSCgo™ Adipogenic Differentiation Medium. Briefly, MSCs were seeded into 24-well plate pre-coated with MSC attachment solution at 6 × 10⁴ cells/well (3 × 10⁴/cm²) in basal medium. After 24 hours or when the cells had reached 80% confluency, the medium was exchanged to differentiation medium containing Incucyte® Opti-Green and BioTracker 488 Green Lipid Droplet Dye. Following 12 days of culture, the medium was exchanged to complete basal medium, and the cells were cultured for a further 4 days. Throughout differentiation, high-definition phase-contrast and green fluorescence images were acquired using the Incucyte® System and analyzed using integrated software. Label-free quantification was performed using the Incucyte® AI Cell Health Analysis Software Module, which enables AI-based segmentation of heterogenous cell morphologies to give univariate morphology metrics (AI Cell Segmentation), Surface Marker Expression At regular time points throughout differentiation, cells were stained for viability and with the MSC marker panel (CD34, CD45, CD73, CD90, and CD105) as well as specific lineage differentiation markers. Briefly, following harvest using trypsin, cells were washed in PBS and stained for viability using Zombie Violet™ for 20 minutes. The cells were seeded into a 96-well V-bottom plate at 20K/well and washed twice with PBS + 2% FBS before staining with the MSC marker antibody cocktail as described in Table 2 for 30 minutes at room temperature. The cells were washed twice by centrifugation (5 min at 1,000 × g) using PBS + 2% FBS. Cells were then resuspended in 20 µl of PBS + 2% FBS and analyzed on the iQue® HTS cytometer. For ALPL, a surface marker, live cells were stained with the ALPL antibody in parallel to the marker panel. For intracellular Fatty Acid Binding Protein 4 (FABP4) staining, cells were fixed using fixation buffer for 20 minutes at room temperature, washed twice in permeabilization buffer, and then stained with FABP4 antibody in PBS + 2% FBS for 30 minutes at room temperature. Cells were washed twice by centrifugation before being analyzed using the iQue® Platform. Data was analyzed using integrated iQue Forecyt® Software. Results Real-Time Quantification of Osteogenic Differentiation A key indicator of MSC quality is the ability to differentiate into cells of multiple lineages, including osteoblasts. Osteogenic differentiation is typically evaluated using an endpoint histological stain for mineralized calcium deposits such as Von Kossa or Alizarin Red S staining. However, these differentiation experiments can typically last for 21 days before the differentiation can be confirmed, the staining process can be time consuming and labour-intensive, they require destruction of the cell samples, and they are typically only semi-quantitative without further laborious analysis steps. A live-cell solution would allow quicker confirmation of the differentiation potential, insight into the dynamics of the differentiation process, and preservation of precious cellular material for further evaluation including surface marker analysis. Here, we demonstrate a simple assay for live-cell quantification of osteogenic differentiation in real-time. Calcein (not to be confused with the viability stain calcein AM) binds to the calcium in growing calcium phosphate and calcium carbonate crystals, which are extracellularly deposited as cells are directed to the bone lineage. As calcein can be incubated with cells for the duration of an experiment, it can be used for continuous assessment of mineralization, and in 3D cultures has been used as a superior alternative to Alizarin Red.⁵ B Calcein (Total area) x10⁵ Time (days) 0 5 10 15 20 0 2 4 6 8 10 12 14 16 18 20 Basal medium Osteogenic Rapid osteogenic Basal medium Osteogenic Rapid Osteogenic 0d 0d 0d 0d 0d 0d 2d 2d 2d 2d 3d 3d 10d 10d 10d 10d 9d 9d A 5 Figure 1: Fluorescent Evaluation of Osteogenic Differentiation of MSCs Cultured in Osteogenic Differentiation Medium Using Live-Cell Analysis. (A) Representative fluorescence and phase-contrast images of MSCs cultured in osteogenic medium, rapid osteogenic medium or MSC basal medium with calcein at intervals during a 21-day period. (B) Quantification of fluorescent calcein signal from images captured during the differentiation period. A CD90 CD73 CD105 CD45 CD34 0 20 40 60 80 100 120 Marker Percentage positive (%) Day 2 Day 4 Day 6 Day 9 Day 11 Day 17 B 2 4 6 9 11 13 16 17 21 0 20 40 60 80 100 ALPL Time (days) Percentage positive (%) Basal medium Osteogenic Rapid osteogenic 6 Figure 1A demonstrates that osteogenic differentiation can be fluorescently detected as early as 2 days, as indicated by the appearance of green fluorescent deposits within the culture at that time point. In contrast, no fluorescent signal was observed in the control condition. There was also a visual difference in the morphology of cells cultured in osteogenic medium compared to those in basal medium, although it was more subtle and the mineral deposits were only distinct at the later time points. Thus, the calcein stain allows a robust, non-perturbing and clear early detection method for evaluating osteogenic differentiation. Rapid osteogenic medium offers faster differentiation of osteoblasts, with a differentiation timescale of 15 days rather than 21 days. Similar to the osteogenic medium, calcium fluorescence was observable in cells cultured in the rapid differentiation medium 3 days post-culture initiation (Figure 1A). The mineral deposition was visible by eye after 6 days, and the pattern of deposition was slightly more regular than in the osteogenic medium. Figure 1B depicts the quantification of this fluorescent signal throughout the differentiation period. In cells cultured in osteogenic media, there was an evident increase in the total fluorescent area beginning at 48 hours post-culture initiation, rapidly reaching a plateau which was broadly maintained for the remaining experimental time course. The peaks and slow decreases in the fluorescent signal reflect the replenishment of the calcein dye during media changes followed by a small amount of bleaching due to the fluorescent imaging. Differentiation as indicated by calcein fluorescence could also be easily quantified for cells in rapid osteogenic medium, with a significant rise following the change to rapid osteogenic medium, reaching a plateau that was maintained for the remainder of the experiment, with less variation arising from media replenishment. Figure 2: Expression of MSC and Osteogenic Differentiation Markers During Culture With Osteogenic Differentiation Medium as Evaluated Using HTS by Cytometry. (A) Expression of markers of MSC identity when MSCs are cultured in rapid osteogenic medium. (B) Quantification of alkaline phosphatase (ALPL) expression as an indicator of osteogenic differentiation. A 0d 3d 9d B 0 2 4 6 8 10 1 2 14 16 0 5 10 15 Time (days) Lipid dye (Total Area) x105 Adipogenic Basal medium 7 Osteogenic differentiation can also be confirmed and quantified by investigating cellular identity using cell surface marker expression. MSC identity, representing a multipotent, self-renewing population, is typically defined as positive expression of CD90, CD73, and CD105, and lack of expression of CD45 and CD34. Figure 2 shows the changes in cellular marker expression during the differentiation period using rapid osteogenic media. The decreases in positive markers and increase in negative markers indicate a reduction in the self-renewing MSC population as the differentiation progressed. ALPL is expressed at a low level in undifferentiated MSCs. However, during the experiment there was a clear increase of ALPL in the cells cultured in osteogenic medium and rapid osteogenic medium compared to basal medium, indicating that the cells were directed to the desired osteoblastic cellular identity (Figure 2B). In rapid osteogenic medium, ALPL expression was markedly increased above the control, rising from around 25% positive at day 2 to around 70% at day 17, indicating a substantial population of maturing osteoblasts. ALPL is primarily expressed by osteoblasts during the early stages of osteogenic differentiation and bone formation. It is a marker of osteoblast activity and is involved in the mineralization process. The reduction in ALPL towards the end of the experiment with osteogenic medium may be indicative of the osteoblast maturation into osteocytes, which arise once osteoblasts are surrounded by their own matrix. Once mature osteocytes become embedded in the bone matrix, the expression of ALPL significantly decreases.⁶ Real-Time Quantification of Adipogenic Differentiation Adipogenic differentiation, similarly to osteogenic, is usually detected using a destructive, endpoint histological stain such as Oil Red O. These methods are semi-quantitative, have low sensitivity for early differentiation, and are labor intensive. A live-cell analysis approach is a desirable alternative with obvious benefits. Here, a fluorescent lipid dye was employed for the early detection of adipogenesis through labeling of the characteristic formation of lipid droplets within the cells. Figure 3: Fluorescent Evaluation of Adipogenic Differentiation of MSCs Cultured in Adipogenic Differentiation Medium Using Live-Cell Analysis. (A) Representative fluorescence and phase-contrast images of MSCs cultured either in adipogenic medium or basal medium with fluorescent lipid dye, acquired at intervals during a 15-day period. (B) Quantification of fluorescent lipid signal from images acquired during the differentiation period. Figure 4: Analysis of Morphological Changes Accompanying Adipogenic Differentiation of MSCs Cultured in Adipogenic Differentiation Medium Using Live-Cell Analysis. (A) Zoomed phase-contrast images of cells during the adipogenic differentiation process. (B) Representative phase-contrast images of cells with overlaid AI Cell Segmentation mask (yellow outlines). (C) Average phase object area at different time points. (D) Average object eccentricity at different time points. A B C 0 3 7 10 13 0.75 0.80 0.85 0.90 Time (days) Average Object Eccentricity 0 3 7 10 13 0 1000 2000 3000 4000 Time (days) Avg Phase Object Area (µm²) D 0d 0h 0m 6d 0h 0m 10d 0h 0m 0d 0h 0m 6d 0h 0m 10d 0h 0m 8 The formation of lipid droplets within the differentiating MSCs is distinctly visible in the images in Figure 3A. The green fluorescent signal was detectable by day 3 following culture initiation in the cells cultured with adipogenic medium, with very low staining in the control basal medium MSCs. Adipocyte morphology with internal lipid droplets can be observed in the phase images, co-localized to the droplet stains. The quantification of the lipid signal is presented in Figure 5B. For the duration of the experiment lipid staining in basal medium was negligible, in marked contrast to the adipogenic media samples, which showed a larger total fluorescent area from day 3 following the start of the differentiation process. The peaks and slow decreases in fluorescent staining here correspond to the replenishment and bleaching of the stain, similar to the calcein. Figure 5: Expression of MSC and Adipogenic Differentiation Markers During Culture With Adipogenic Medium as Evaluated Using HTS by Cytometry. (A) Expression of markers of MSC identity. (B) Quantification of fluorescent lipid dye and FABP4 as indicators of adipogenic differentiation. A Percentage positive (%) CD90 CD73 CD105 CD45 CD34 0 20 40 60 80 100 120 Marker P erc entag e p ositiv e (%) Day 4 Day 6 Day 9 Day 11 Day 17 Day 2 120 100 80 60 40 20 0 CD90 CD73 CD105 CD45 CD34 Day 2 Day 4 Day 6 Day 9 Day 11 Day 17 Marker B Percentage positive (%) 2 4 6 9 11 17 0 20 40 60 80 100 Lipid Time (days) P ercentag e p ositive (%) Adipogenic Basal medium 100 80 60 40 20 0 2 4 6 9 11 14 Time (days) Lipid Percentage positive (%) 2 4 6 9 11 17 0 20 40 60 80 100 FABP4 Time (days) P ercentag e p ositive (%) Adipogenic Basal medium 100 80 60 40 20 0 2 4 6 9 11 14 Time (days) FABP4 9 In addition to employing fluorescent labeling of lipid droplets to quantify adipocyte presence, their distinct morphology Figure 4A allows a label-free approach to quantifying adipogenesis. Figure 4 depicts how AI-based cell segmentation can be used to evaluate key cell metrics such as area and eccentricity on a population level and is able to accurately segment low-contrast cells with heterogenous morphologies until confluency is reached (Figure 4B). As adipogenic differentiation progresses, cell morphology changes from flattened, spindle-shaped cells to a greater proportion of the cell population having a rounded or oval shape. This is reflected in Figure 4C and D, with both area and eccentricity initially high and decreasing as the time course progresses. These two metrics, in combination with visual assessment of the images, can be used to confirm adipogenic differentiation without requiring the addition of fluorescent reagents. When examining expression of markers during adipogenic differentiation, a distinct change in cellular identity was observed during the process (Figure 5). As in the osteogenic differentiation, expression of positive MSC markers remained high for the duration of the experiment. However, both negative markers, CD34 and CD45, were elevated from 4 days and higher levels were maintained until the adipocytes became more mature. The intracellular lipid dye can be detected using single cell cytometry in addition to fluorescence imaging. By day 4 of the process, 50% of the cells contained the dye, compared to 0% in the basal medium control, which is a clear early confirmation of adipogenesis. Concurrently, FABP4 expression was elevated in the adipogenic medium from day 4 of the differentiation process. Specifications subject to change without notice. © Sartorius Lab Instruments GmbH & Co. KG. Status: 09 | 2024 North America Sartorius Corporation 565 Johnson Avenue Bohemia, NY 11716 USA Phone +1 734 769 1600 Email: orders.US07@sartorius.com Europe Sartorius UK Ltd Longmead Business Centre Blenheim Road Epsom Surrey, KT19 9QQ United Kingdom Phone +44 1763 227400 Email: euordersUK03@sartorius.com Asia Pacific Sartorius Japan K.K. 4th Floor, Daiwa Shinagawa North Bldg. 1-8-11, Kita-Shinagawa 1-chome Shingawa-Ku Tokyo 140-0001 Japan Phone +813 6478 5202 Email: euordersUS07@sartorius.com Find out more: sartorius.com/incucyte sartorius.com/hts For questions, email: AskAScientist@sartorius.com Summary and Outlook The use of advanced analysis technologies has significantly enhanced the ability to assess MSC differentiation in real-time, providing valuable insights into the dynamics of the differentiation process. The successful application of these technologies in both osteogenic and adipogenic differentiation demonstrates their versatility and effectiveness. By providing a comprehensive view of the cellular changes, these methods pave the way for more standardized, reliable, and reproducible assessments of MSC functionality. These methods offer several advantages over traditional endpoint assays, including quicker confirmation of differentiation potential, preservation of cellular material for further analysis, and the ability to monitor multiple parameters simultaneously. By adopting these advanced technologies, researchers can improve the efficiency and depth of MSC research, ultimately enhancing the therapeutic potential of MSCs for treating various diseases and conditions. References 1. Lee, D.H., Kim, S.J and Kim, S.A. et al. Past, present, and future of cartilage restoration: from localized defect to arthritis. Knee Surgery & Related Research. 2022. Vol. 34, 1. 2. Kurtzberg J, Prockop S, Teira P, Bittencourt H, Lewis V, Chan KW, Horn B, Yu L, Talano JA, Nemecek E, Mills CR, Chaudhury S. Allogeneic human mesenchymal stem cell therapy (remestemcel-L, Prochymal) as a rescue agent for severe refractory acute graft-versus-host disease in pediatric patients. Biol Blood Marrow Transplant. 2014. Vol. Feb, 20(2). 229-35. 3. Yoshihisa Yamada, Shingo Minatoguchi, Hiromitsu Kanamori, Atsushi Mikami, Hiroyuki Okura, Mari Dezawa, Shinya Minatoguchi. Stem cell therapy for acute myocardial infarction – focusing on the comparison between Muse cells and mesenchymal stem cells. Journal of Cardiology. 2022. Vol. 80, 1. 80-87. 4. Dominici, M., et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006. Vol. 8, 4. 315-317. 5. White, Kristopher, et al. Calcein Binding to Assess Mineralization in Hydrogel Microspheres. Polymers. Basel: s.n., 2021. Vol. Jul 11, 13(14). 2274. 6. Franz-Odendaal, Tamara A, Hall, Brian K and Witten, P Eckhard. Buried alive: How osteoblasts become osteocytes. Developmental Dynamics. 2006. Vol. Jan 235, 1. 176-90.