Human mesenchymal stem cell expansion on laminin-521 in serum-free and xeno-free culture conditions

Abstract

Laminin-521 (LN521) is a crucial adhesion protein found in natural stem cell niches and plays an important role in maintaining human pluripotent stem cell (PSC) properties. This study aimed to investigate the effects of LN521 on human umbilical cord-derived mesenchymal stem cell (UC-MSC) characteristics in Serum-free and Xeno-free culture conditions as a step toward clinical application. In our experiment isolated UC-MSC via explant method were expanded as a homogeneous monolayer and morphologically, presented typical MSC-like morphology (spindle-shaped) from passage three to six when cultured on either LN521 or CELLstart™. Almost, 90% confluency was reached after 4 days of culture with an EI of approximately 11.2 with no statistically significant differences on LN521 and CELLstart™ in all six passages. Phenotypic characterization of UC-MSC cultured on LN521 or CELLstart™ using flow cytometry, along with the expression of the same biomarkers in gene level analyzed by quantitative reversed transcription revealed identical CD73, CD90, CD105, CD34, CD45, CD19, CD14, and HLA-DR expression pattern at passages three and six in both LN521 and CELLstart™. Moreover, UC-MSC cultured in the presence of LN521 and CELLstart™ showed the same adipogenesis, chondrogenesis and osteogenesis differentiation potential, and normal chromosome structure highlighting genetic stability. Ultimately, LN521 is comparable to CELLstart™ in supporting UC-MSC expansion and maintaining their characteristics in serum-free and xeno-free culture conditions.

Introduction

Mesenchymal stem cells (MSCs) have appeared as an outstanding promise in the field of regenerative medicine for having a stable genome and the ability to self-renew and differentiate to numerous cell lineages when proper culture conditions are provided. ¹ Besides these characteristics, MSC has the potential of homing to the sites of injury and show an immunomodulatory effect by stimulating the production of trophic factors in large amounts.^2–7 All these aspects make MSCs a promising therapeutic option to approach many pathological conditions such as, graft versus host disease (GvHD), ⁸ lung injury,^9,10 diabetes,^11,12 stroke, ¹³ and injury to spinal cord. ¹⁴ On the other hand, there is another advantage of the MSCs that makes it more popular for clinical usage which is having less ethical conflicts as compared to other types of stem cells for instance the embryonic stem cells. ¹⁵

The first clinical trial using MSCs was done in 1995, by injecting the cultured MSCs to check the safety of this type of therapy. ¹⁶ The first conditional approval for MSCs therapy was approved in 2012 in New Zealand and Canada. They used MSCs to handle Graft versus Host Disease (GvHD). Afterward, this method got approval for Crohn’s Disease-related fistula treatment in Japan.^17,18 According to the report in July 2023, more than 3500 ongoing clinical trials and 10,000 registered trials evaluated the therapeutic capabilities and characteristics of MSCs.^19,20

The existence of MSCs in various types of tissues in the human body including, bone morrow (BM), adipose tissue (AT), and umbilical cord (UC) is another point that makes it more available and as a result more accepted for therapeutic purposes in comparison to other types of stem cells. ²¹ Nevertheless, the low number of MSCs derived out of these sources is insufficient for clinical application. Thus, for having an effective MSCs transplantation, an appropriate number of MSCs is required, which necessitates the expansion of isolated MSCs in vitro.

To improve efficient stem cell therapy, the MSCs propagation process, should be done under protected, appropriate, and applicable methods which maintain the properties of these cells. ²² Reaching this goal necessitates the large-scale standardized expansion of these cells under good manufacturing practice (GMP) conditions. One of the most important steps for achieving this standardization in clinical therapeutic goals is optimal culture condition identification. Furthermore, prevention of using animal-derived culture components for MSCs expansion is mandatory to guarantee the safety of using MSCs for therapeutic uses. For instance, fetal bovine serum (FBS) was broadly used in MSCs expansion for clinical protocols that developed safety concerns such as enhancing the risk of animal-derived antigens transmission to the MSCs recipient. ²³ This brought the idea of using culture conditions devoid of any animal products to confirm the safety of the patients after transplantation. Accordingly, xeno-free culture conditions were considered as a requirement for the clinical application of human MSCs. Formerly, researchers have shown the appropriateness of serum-free and xeno-free culture conditions in stabilizing and improving the human induced pluripotent stem cells, ²⁴ and human embryonic stem cells ²⁵ expansion in vitro. However, regarding MSCs, investigations are not consistent about the effect of xeno-free culture condition on the MSCs. Studies demonstrated variation in the influence of xeno-free culture conditions on MSCs prior to use for clinical application. ²⁶

Moreover, the critical role of the microenvironment in MSCs properties maintenance is undeniable. Thus, stem cell microenvironment and niche should be retained while culturing these cells in vitro. ²⁷ To overcome this, many researchers worked on culturing MSCs on their extracellular matrix components.^28–30 Utilizing extracellular matrix for MSCs xeno-free culture showed positive influences on preservation of the MSCs properties. CELLstart™ is one of the recommended and approved extracellular matrix using as a coating material for expanding the MSCs in xeno-free medium. Moreover, it is shown that this method facilitates controlling the MSCs differentiation potentials. ³¹

Extracellular matrix contains collagen, growth factors, adhesion proteins, proteoglycans, and laminins.^32–34 Laminins are widely expressed in human bone marrow and manufactured by the bone marrow stromal cells. ³⁵ Therefore, laminins are considered as a part of the MSCs niche. This study aims to investigate the effect of Laminin-521 (LN521) on human UC-MSCs characteristics in a serum-free and xeno-free culture condition.

Materials and methods

Ethics statement

This project was approved by the medical research committee at the College of Medicine and health sciences, Sultan Qaboos University (Ref. No, SQU-EC/327/2021). Two umbilical cord samples were collected after normal deliveries at Sultan Qaboos Hospital (Muscat, Oman) in November 2023. Informed consent was signed by their mothers.

Umbilical cord dissection and culture

Explant and enzymatic methods are two main procedures to isolate UC-MSC. ³⁶ In this study, we used the explant method by cutting the umbilical cord (UC) into sections of a few centimeters that is called explants. The UC with a length of 15 cm were collected in DMEM (100 U/mL penicillin and 100 µg/mL streptomycin) (Gibco, New York, USA). The UC was washed with 70% ethanol then twice with phosphate-buffered saline then cut into 2–3 mm pieces with sterilized scissors and forceps to remove the vein and arteries. Next, UC pieces were cut into smaller pieces. Then, explants were divided into three 60 mm petri dishes coated with CELLstart™ substrate (Gibco, New York, USA) in the StemPro^® MSC SFM complete Medium (Gibco, New York, USA) (StemPro^® MSC SFM Basal Medium plus StemPro^® MSC SFM Xeno-free Supplement) added with 50 µg/mL Gentamicin, and 2.5% AB-Human Serum (Gibco, New York, USA). Explants were left undisturbed for 7 days at 37°C in a 5% CO2 incubator. After 7 days, media were changed every 3 days. After growing the UC-MSC out from tissue pieces, UC explants were removed. During the cell culture period, the morphology of isolated and expanded MSC were checked using inverted microscope. For subculturing the UC-MSC, cells were detached using CTS™ TrypLE™ (Gibco, New York, USA). Then cells were collected and washed with StemPro^® MSC SFM complete Medium through centrifugation at 100–200 × g for 5 min.

Coating process with CST™ CELLstart^™ substrate and laminin-521 (LN521)

First, the CTS™ CELLstart™ were diluted and mixed gently in Dulbecco’s Phosphate Buffered Saline (DPBS) CTS™ with calcium and magnesium (Gibco, New York, US) with the ratio of 1:100. Next, the CTS™ CELLstart™ Substrate solution was added to each cell culture vessels and were incubate at 37°C in a humidified atmosphere of 5% CO2 in air for 60–120 min according to the manufacturer’s instructions. Finally, CTS™ CELLstart™ Substrate solutions were removed immediately before use and were replaced with StemPro^® MSC SFM complete Medium.

The optimum concentration of LN521 (human recombinant laminin 521, Biolamina, Sweden) for coating (10 μg/mL) were prepared by diluting Biolaminin stock solution in 1xDPBS with calcium and magnesium. Then plates were coated with diluted LN521 and were incubated at 4°C overnight according to the manufacturer’s instructions.

Proliferation and expansion index

Expansion Index (EI) of MSC were performed using the trypan blue exclusion cell count method. In brief, MSC were plated into 12-well plate coated with CELLstart™ and LN521, at 3 × 10⁴ MSC/well (~8000 cells per cm²) in serum-free and xeno-free medium. Cells were harvested from 3 wells every day till day 4 and were counted on a hemocytometer using Trypan Blue. To calculate the EI, the number of cells at the time point of interest was divided into the number of cells at day 0. For each time point cells were cultured in triple.

Surface antigen expression pattern analysis by flow cytometry

Phenotypic characterization of UC-MSC were done by surface markers detection using flow cytometry. Cells cultured with CELLstart™ and LN521 were detached and collected at passage three and passage six by CTS™ TrypLE™ Select Enzyme. A suspension of 1 × 10⁵ cells in 100 µL in PBS containing 2% FBS was added to each eppendorf tube. Fluorescently labeled anti-human-antibodies [phycoerythrin (PE)-conjugated antibodies] (Becton Dickinson, San Jose, CA, USA), against CD14, CD19, CD34, CD45, HLA-DR, and CD73, CD90, CD105, were used to stain the cells. After incubating the cells for 30 min at 4°C in the dark, and washing with PBS containing 2% FBS, they were immediately run by BD LSRFortessa™ Cell Analyzer - Flow Cytometers (Becton Dickinson, New Jersey, USA). For data analysis, FlowJo v-10 software was used. Unstained cells and isotype negative controls were used to optimize the voltage and color compensation.

Gene expression pattern analysis after expansion via quantitative reverse transcription qRT-PCR

UC-MSCs were seeded in 60 mm Petri dishes coated with CELLstart™ and LN521 with 1 × 10⁴ viable cells/cm2 density in StemPro^® MSC SFM. Next, UC-MSCs were harvested from passages three and six after reaching 70%–80% confluency. The total RNA was extracted using an RNA extraction kit (Invitrogen, USA). The concentration and purity of RNA were checked by a nanodrop spectrometer. Then, cDNA was produced from the extracted RNA using the High-Capacity RNA-to-cDNA Kit (Gibco, New York, USA). A thermal cycler PCR machine (Applied Biosystem, Massachusetts, USA) was used for PCR amplification. Later, expression of THYI (CD90), NT5E (CD73), ENG (CD105), CD34, CD14, HLA-DR, PTPRC (CD45) were investigated by RT-qPCR using TaqMan Universal PCR Master Mix (Applied Biosystem, Massachusetts, USA) with TaqMan probes (Applied Biosystem, Massachusetts, USA) according to the manufacturer’s protocol.

Quantitative reverse transcription PCR (RT-qPCR) data analysis

The ∆∆ Ct method was used to calculate fold differences in gene expression by RT-qPCR. GAPDH was used as an internal control to normalize the data for each gene. No template control (NTC) was placed with each reaction set up to check for contamination in reaction components if present. All the samples were run in triplicates.

Multi-lineage differentiation potential analysis

To analyze UC-MSC multi-lineage differentiation potential, cells expanded for six passages were seeded in 12-well plate coated with LN521 and CELLstart™ with the density of 1 × 10⁴ viable cells/cm² in StemPro^® MSC SFM (Gibco, New York, US) and were incubated in 37°C in a humidified atmosphere at 5% CO₂. After 24 h, media were replaced with pre-warmed Complete Adipogenesis Differentiation Medium, Complete Osteogenesis Differentiation Medium, and Complete Chondrogenic Differentiation Medium for Adipogenic, Osteogenic and Chondrogenic differentiation respectively, and incubation was continued (Media were changed every 3 days).

Adipogenic differentiation

Essentially, UC-MSC continued to undergo limited expansion as they differentiated under adipogenic conditions. At day 21, adipogenic differentiation media were removed, and wells were rinsed once with DPBS. So, cells were fixed with 4% formaldehyde solution for 30 min, then rinsed twice with DPBS. Subsequently, Oil Red O stain was applied, and cells were incubated for 15–30 min and were rinsed twice with DPBS. Cells were visualized under the light microscope.

Osteogenic differentiation

In osteogenic differentiation, media were removed from a 12-well plate and rinsed once with DPBS after 21 days of culture. After that, cells were fixed with 4% formaldehyde solution for 30 min, rinsed twice with distilled water and stained with 2% Alizarin Red S solution (pH 4.2) for 2–3 min. Finally, wells were rinsed three times with distilled water and visualized under the light microscope.

Chondrogenic differentiation

After 21 days of culturing expanded MSC from passage six in a chondrogenic differentiation culture, media were removed from the culture vessel and rinsed once with DPBS. Cells were fixed with a 4% formaldehyde solution for 30 min. After fixation, wells were rinsed with DPBS and stained with 1% Alcian Blue solution prepared in 0.1 N HCL for 30 min. Wells were rinsed three times with 0.1 N HCl, and distilled water was added to neutralize the acidity. Cells were visualized under the light microscope.

Gene expression pattern analysis after differentiation

With the aim of gene expression pattern analysis, UC-MSC from passage six were seeded in a 12-well plate coated with LN521 and CELLstart™ with the density of 1 × 10⁴ viable cells/cm² in StemPro^® MSC SFM. Later, StemPro^® MSC SFM media were replaced with pre-warmed Complete Adipogenesis, Osteogenesis, and Chondrogenic Differentiation Medium after 24 h (media were changed every 3 days). After 21 days, UC-MSCs were harvested, RNA was extracted, and cDNA was generated to proceed with the gene expression analysis by Quantitative real-time PCR (qPCR) analysis. The expression of aggrecan (ACAN), bone gamma-carboxyglutamate protein (BGLAP), and adiponectin (ADIPOQ) was analyzed by RT-qPCR using TaqMan Universal PCR Master Mix with TaqMan probes according to the manufacturer’s protocol to investigate the chondrogenic, osteogenic, and adaptogenic differentiation of MSC respectively. GAPDH was used as an internal control to normalize the data for each gene. A real-time reaction with no template genes was used as a negative control. The Ct method was used to calculate the fold difference in gene expression (Samples were run in triplicates).

Karyotyping

To analyze the effect of LN521 and CELLstart™ on the genetic stability of the UC-MSC after six passages, UC-MSC were seeded with the density of 3 × 10⁴/cm² in 60 mm Petri dishes. After reaching 70%–80% confluency, 30 µL of colcemid (10 µg/mL) was added to the Petri dishes and incubated at 37°C for 45 min. Then, cells were detached and collected. Next, a 7 mL mixture of hypotonic solution (0.075M KCL + 1% Sodium citrate [6:4 ratio]) was added and incubated at 37°C for 12 min. After centrifugation, 10 mL fixative (3:1 methanol: acetic acid) was added and incubated overnight in the refrigerator. The following day, 25–35 µL of cell suspension was dropped on a precleaned cold slide at optimum temperature and humidity and allowed to dry at 60°C overnight. After the G-banding procedure, 30 metaphases were examined, for numerical or structural abnormalities, distributed equally across at least two independently established cultures.

Statistical analysis

A descriptive analysis was performed for all variables in this study. Comparisons for all pairs were performed using the paired T-test. Statistical significance was defined by a p-value of 0.05 or less for all tests. All data were analyzed using SPSS version 23.

Results

UC-MSCs morphology

After incubating the explants on CELLstart^™ substrate-coated 60 mm Petri dishes in the StemPro^® MSC SFM complete Medium supplemented with 2.5% AB-Human Serum, the fibroblastic-like MSCs were grown out of the tissue pieces and attached to the vessels surface at day 14. Cells reached 50% and 80%–90% confluency on days 20 and 25, respectively. In this study, MSCs were isolated from two, different umbilical cord units. Based on our observation, UC-MSC morphologically sustained a stable spindle shape following six passages. Figure 1 shows the UC-MSC morphology at passages three and six derived from two different umbilical cord, respectively (Figure 1).

Figure 1.

UC-MSC morphology at passage three and six derived from two different umbilical cord. UC-MSC expanded on LN521 and CELLstart™ using serum-free and xeno-free medium showed typical MSC-like morphology following six passages. (C = CELLstart™, L = Laminin-521, SQU.1 = MSCs from UC sample number 1, SQU.2 = MSCs from UC sample number 2, P = Passage).

UC-MSCs proliferation and expansion index

Consequently, expanded UC-MSCs from passage zero to passage six using serum-free and xeno-free condition showed no statistically significant differences in EI while using LN521 and CELLstart™. In our study, UC-MSCs reached EI of approximately 1.4, 2.8, 5.4, and 11.2 with 90% confluency at day one, two, three and four of culture on LN521 and CELLstart™ using serum-free and xeno-free medium, respectively. Figure 2 shows UC-MSCs expansion index at passage three and six for 4 days cultivated on LN521 and CELLstart™ using serum-free and xeno-free medium from two umbilical cords (Figure 2).

Figure 2.

UC-MSCs expansion index at passage three and six cultivated on LN521 and CELLstart™ using serum-free and xeno-free medium for 4 days (observations were obtained in triplicate and counting the cells were done three times for each time point for SQU.1 = MSCs from UC sample number 1, and SQU.2 = MSCs from UC sample number 2).

Surface antigen expression pattern analysis by flow cytometry

The surface antigen expression pattern of UC-MSCs, analyzed by flow cytometer, demonstrated the stable expression of biomarkers following six passages. Based on our result, the expressions of CD73, CD90, and CD105 on UC-MSCs at passage three and passage six were positive (for more than 95% of the MSCs population), but the expression of CD14, CD19, CD34, CD45, and HLA-DR was negative (for more than 98% of MSCs population). Immunophenotyping results presented identical characteristics at passage three and six of UC-MSCs expanded on LN521 and CELLstart™ using serum-free and xeno-free medium. Figure 3 shows the percentage of surface antigen expression by expanded UC-MSCs on LN521 and CELLstart™ using serum-free and xeno-free medium at passages three and six from two umbilical cords by flow cytometry (Figure 3). The expressions pattern of CD73, CD90 and CD105, CD14, CD19, CD34, CD45, and HLA-DR on UC-MSC at passage three and passage six were shown in Supplemental Figure.

Figure 3.

Percentage of surface antigen expressed by expanded UC-MSCs on LN521 and CELLstart™ using serum-free and xeno-free medium at passage three and six by Flow Cytometry. More than 95% of MSCs population expressed CD73, CD90 and CD105, and less than 2% of MSCs population expressed CD14, CD19, CD34, CD45, and HLA-DR. (observations were obtained in triplicate from two different umbilical cords) (Results are the mean percentage of antigen expression from SQU.1 = UC sample number 1, and SQU.2 = UC sample number 2).

Gene expression pattern analysis after expansion via quantitative reverse transcription qRT-PCR

The UC-MSCs gene expression pattern analysis by quantitative reversed transcription qRT-PCR, showed no statistically significant changes in expression of our target genes [THYI (CD90), NT5E (CD73), ENG (CD105), CD34, CD14, HLA-DR, PTPRC (CD45)] at passage three and six after expanding on LN521 and CELLstart™ using serum-free and xeno-free medium. Figure 4 shows the expression pattern of THYI, NT5E, ENG, CD34, CD14, HLA-DR, PTPRC on UC-MSCs at passage three and six by qRT-PCR (Figure 4).

Figure 4.

Expression pattern of THYI, NT5E, ENG, CD34, CD14, HLA-DR, PTPRC on UC-MSC expanded on LN521 and CELLstart™ using serum-free and xeno-free medium, at passage three and six by qRT-PCR (Experiments were performed in triplicate) (C = CELLstart™, L = Laminin-521, SQU.1 = MSCs from UC sample number 1, SQU.2 = MSCs from UC sample number 2, P = Passage).

Multi-lineage differentiation potential analysis

The multilineage differentiation capability of UC-MSCs expanded on LN521 or CELLstart™ using serum-free and xeno-free medium at passage six was investigated based on their adipogenesis, osteogenesis, and chondrogenesis differentiation potentials. This study explored adipogenesis under the microscope by observing the accumulated lipid drops after oil-red O staining after 21 days of differentiation. MSC showed low density and fibroblast-like morphology at day zero regarding the osteogenic differentiation process. After 21 days of differentiation in complete osteogenic differentiation media, the morphology of the cells was changed to a cuboidal shape, and the density of the cells was increased. Also, Alizarin Red staining showed a positive result. The chondrogenesis process was examined by observing elongated or round spheres of chondrocytes, which were positive for Alcian Blue staining. Mesenchymal stem cells cultured in StemPro^® MSC SFM media as a control group showed no morphological changes or positivity in staining with Oil red O, Alizarin Red, and Alcian Blue staining. Our investigation through differentiation assay showed the same adipogenesis, chondrogenesis and osteogenesis potential in UC-MSCs at passage six expanded in LN521 and CELLstart™ using serum-free and xeno-free medium and differentiated by complete adipogenesis, osteogenesis, and chondrogenic differentiation medium. Figure 5 shows the multilineage differentiation potential of UC-MSC after six passages cultured on LN521 and CELLstart™ (Figure 5).

Figure 5.

Multilineage differentiation potential of expanded UC-MSCs for six passages on LN521 and CELLstart™ using serum-free and xeno-free medium and differentiated by complete adipogenesis, osteogenesis, and chondrogenic differentiation medium. Our results showed the identical adipogenesis (a), chondrogenesis (b), and osteogenesis (c) differentiation potential of expanded UC-MSCs cultured on LN521 or CELLstart™ (Experiments were performed in triplicate) (C = CELLstart™, L = Laminin-521, SQU.1 = MSCs from UC sample number 1, SQU.2 = MSCs from UC sample number 2, P = Passage, 10x and 40x = Magnification).

Gene expression pattern analysis after differentiation

Quantitative real-time PCR analysis was done to investigate the expression pattern of differentiation-related genes in UC-MSCs from passage six, after differentiation for 21 days in Complete Adipogenesis Differentiation Medium, Complete Osteogenesis Differentiation Medium, and Complete Chondrogenic Differentiation Medium for Adipogenic, Osteogenic and Chondrogenic differentiation in 12-well plate coated with either LN521 or CELLstart™. Our results showed no statistically significant differences in the expression of differentiation-related genes such as adiponectin (ADIPOQ) for adipogenesis, aggrecan (ACAN) for chondrogenesis, and bone gamma-carboxyglutamate protein (BGLAP) for osteogenesis, on differentiated UC-MSCs cultured on LN521 and CELLstart™ (Figure 6).

Figure 6.

Gene expression pattern of expanded UC-MSCs for six passages on LN521 and CELLstart™ using serum-free and xeno-free medium and differentiated by complete adipogenesis, osteogenesis, and chondrogenic differentiation medium. Differentiation related genes showed no statistically significant differences in the presence of LN521 and CELLstart™ (Experiments were performed in triplicate) (C = CELLstart™, L = Laminin-521, SQU.1 = MSCs from UC sample number 1, SQU.2 = MSCs from UC sample number 2, P =Passage).

Karyotyping

UC-MSCs from passage 6 cultured on LN521 and CELLstart™ were arrested in the metaphase stage following treatment with colcemid. A normal karyotype with 46 chromosomes and no obvious cytogenetics abnormalities were observed after analysis of 30 metaphase cells. In addition, no abnormal changes in chromosome structure were observed after culturing cells on LN521 and CELLstart™. Figure 7 shows the genetic evaluation of expanded UC-MSCs using LN521 and CELLstart™ at passage six by karyotyping analysis (Figure 7).

Figure 7.

Genetic evaluation of expanded UC-MSCs on CELLstart™ (a) and LN521 (b) using serum-free and xeno-free medium at passage six by karyotyping analysis (C = CELLstart™, L = Laminin-521, SQU.1 = MSCs from UC sample number 1, SQU.2 = MSCs from UC sample number 2).

Discussion

Achieving a high expansion index while maintaining cell properties is one of the primary goals in stem cell culture with therapeutic applications. Cell expansion is impacted by several factors, including culture media components, atmospheric conditions, and cryopreservation, which result in the complexity of the MSCs manufacturing processes. The ex vivo expanded MSCs using culture media including animal components such as fetal bovine serum (FBS) which has been used for years may possibly restrict the clinical application of these cells through safety concerns. To prevent using animal-derived culture elements for MSCs expansion toward having safer MSCs for therapeutic uses, the replacement of FBS-containing media with serum-free and xeno-free culture condition was suggested. This was supported by studies like Shi et al., research showing the developed expansion rate, higher genetic stability, and lower cellular senescence in serum-free and xeno-free media. ³⁷ In another study reported by Lee et al. MSCs isolated from adipose tissue cultured in serum-free culture condition resulted higher number of cells in a shorter time, developed genetic stability, and reduced cellular senescence in addition to lower immunogenicity, as compared to when FBS containing media were used which offer more secure cell therapy. ³⁸ Extracellular matrix (ECM) is another requirement for MSCs to proliferate and stay undifferentiated. This environment could be mocked through using coating substrates in culture. ³⁸ CELLstart™ is considered the first xeno-free substrate which can be used for the human embryonic stem cells, induced pluripotent stem cells, neural stem cells, and mesenchymal stem cells attachment and expansion without any feeder cells for transition from research to clinic. ³⁹ On the other hand, Laminin is the most important signaling element of all ECM which is responsible for physiologic and structural support of epithelial cells, endothelial cells, muscles, etc. Specifically, Laminin-521 (LN521) subunit is considered as a ubiquitous component for stem cell niches.^40–42 Based on previous studies, LN521 could be a promising coating system for induced pluripotent stem cells (iPSCs) propagation even on a large scale. ⁴²

In this study, we developed a culture condition using serum-free and xeno-free media together with LN521 to derive UC-MSCs. We conducted a comprehensive analysis of the cellular expansion index, morphological changes, differentiation potential, and antigen expression pattern, in addition to the gene expression level and genetic stability of UC-MSCs isolated from two different UC samples cultured in xeno-free and serum-free culture conditions in the presence of two various cell matrices, LN521 and CELLstart™, for six passages.

Throughout checking the characteristic maintenance of UC-MSCs in morphological analysis, we showed expanded UC-MSCs on LN521 and CELLstart™ exhibited typical spindle-shaped morphology after passaging them for six passages. Our data indicated the identical ability of UC-MSCs to propagate on CELLstart™ and LN521 in an optimum seeding density of 8 × 10³ cells per cm². In this optimum seeding density using a 12-well plate, the UC-MSCs achieved the confluency of 90% after 4 days with an EI of approximately 11.2 with no statistically significant difference between LN521 and CELLstart™ conditions. We found a lower cell proliferation rate when using LN521, compared to CELLstart™, at seeding densities lower than 8 cells per cm × 10³ cells per cm². This finding suggests that LN521 may not support cell-cell interaction adequately at low seeding densities. Through expansion protocol optimization, Paccola Mesquita et al. showed the generation of a high number of iPSCs after large-scale expansion even with a lower concentration of LN521. ⁴³ As the cost of cell therapy is one of the most important challenges, the potential of LN521 in supporting iPSC propagation in low concentration shows that LN521 could be considered as also more commercial and prevalent to use for therapeutic purposes.

Other properties of isolated UC-MSCs as antigen expression pattern that were investigated by flow cytometry showed that UC-MSC maintained the expression of CD73, CD90, CD105, and lack of CD34, CD45, CD19, CD14 and HLA-DR, same as MSC characterization criteria proposed by the Cellular Therapy International Society ⁴⁴ over six passages in the presence of LN521 and CELLstart™. In terms of gene expression analysis investigated by quantitative reversed transcription (qRT-PCR), expanded UC-MSC did not display significant differences in the expression pattern of THYI (CD90), NT5E (CD73), ENG (CD105), CD34, CD14, HLA-DR, PTPRC (CD45) at passages three and six when cultured on either LN521 or CELLstart™. Regarding genetic studies, the stable genomes were detected by karyotyping after culturing on LN521 or CELLstart™. Considering all these findings both LN521 and CELLstart™ maintained the fibroblastic-shape morphology, immunophenotype, and expansion potential and the expression of MSC-related biomarkers through antigen expression and gene expression pattern analysis. UC-MSCs expanded using LN521 and CELLstart™, preserved the genomic stability and without altering the chromosome structure or number after sub-culturing in vitro for six passages.

Across our exploration, the capacity of MSCs to differentiate, as analyzed by differentiation assay, showed the equal potential of UC-MSC to differentiate to adipocyte, chondrocyte and osteocyte when cultured on LN521 or CELLstart™. In addition, through qRT-PCR analysis, no statistically significant differences were found in the expression of genes that associated with MSC differentiation, such as aggrecan (ACAN) for chondrogenesis, adiponectin (ADIPOQ) for adipogenesis, and bone gamma-carboxyglutamate protein (BGLAP) for osteogenesis, on LN521 and CELLstart™ matrices. LN521 and CELLstart™ maintained the adipogentic, chondrogenic and osteogenic differentiation potential of UC-MSC. In another study by Gert Vanmarcke et al., through evaluation of various recombinant extracellular matrices for Matrigel replacement to model liver disease, LN521 showed a promising effect on differentiation of human pluripotent stem cell (hPSC) to hepatocyte-like cells. ⁴⁵ In another study, LN521 was applied as a coating system to support the expansion of induced pluripotent stem cells (iPSC). The study reported iPSC with normal morphology, increased proliferation potential, pluripotent state maintenance, karyotype stability, and the capacity to differentiate into the cells of interest.^45,46 The same data were registered earlier by Liu et al. ⁴⁷ The supportive role of LN521 has been shown in other types of cells as well. Albalushi et al. studied the effect of LN521 on human embryonic stem cell lines (hESCs). They described the more stable expression of pluripotency related markers in hESCs for nine passages throughout using LN521. ²⁵ In another study in which LN521 was used for supporting neural cells, Bae et al. showed that LN521 causes neural cell adhesion and survival enhancement. In addition, they found that LN521 can promote the maturation, initial differentiation, and function of these cells. ⁴⁸

(LN521 is commercially available in forms that are GMP-compliant, xeno-free substrate, meeting the stringent requirements for clinical-grade cell culture. Laminin-521 (LN521) aligns closely with regulatory requirements for clinical-grade MSC expansion by providing a GMP-compliant, xeno-free, and chemically defined substrate. By eliminating animal-derived components, LN521 minimizes risks of zoonotic contamination and immunogenicity, while its ability to mimic the native extracellular matrix supports MSC stability, functionality, and therapeutic potential. This defined system ensures reproducibility, scalability for large-scale manufacturing, and compliance with quality control measures necessary for clinical translation

By ensuring safety, consistency, and robust MSC expansion, LN521 provides a standardized platform that enhances the translational potential of MSC-based therapies in regenerative medicine and other clinical applications

Laminin-521 (LN521) offers a unique and highly standardized platform for the clinical-grade expansion of mesenchymal stem cells (MSCs), aligning closely with regulatory requirements and strengthening its potential for therapeutic applications.)

Conclusions

Due to the low number of isolated MSCs, numerous propagation phases are necessary to produce adequate cells while maintaining their properties for clinical applications. The mesenchymal stem cell expansion process is impacted by various culture components, such as serum and cell matrices. This study, found that LN521 plays a supportive role in UC-MSCs expansion, exhibiting comparable morphology, and molecular characteristics to UC-MSCs grown on CELLstart™. Our findings suggest that LN521 could be considered an ideal coating substrate candidate for the UC-MSCs expansion in serum-free and xeno-free culture conditions, contributing to the establishment of a more efficient culture system for the for cell production platforms. While our study provides valuable baseline data and demonstrates the feasibility of the experimental approach, which can serve as a foundation for further investigations, future research including larger number of samples is recommended to investigate the influence of that variability in donor characteristics (e.g. genetic background, gestational age, or health status).

Supplemental Material

sj-jpg-1-jbf-10.1177_22808000251332110 – Supplemental material for Human mesenchymal stem cell expansion on laminin-521 in serum-free and xeno-free culture conditions

Supplemental material, sj-jpg-1-jbf-10.1177_22808000251332110 for Human mesenchymal stem cell expansion on laminin-521 in serum-free and xeno-free culture conditions by Halima Albalushi, Mohadese Boorojerdi, Elias Said, Halima Al Shehhi, Nihal Al Riyami, Mohammed Al Rawahi and Murtadha Al Khabori in Journal of Applied Biomaterials & Functional Materials