Intra-hydrogel culture prevents transformation of mesenchymal stem cells induced by monolayer expansion

Tongmeng Jiangabc, Junting Liub, Yiqiang Ouyangd, Huayu Wue, Li Zheng*abc, Jinmin Zhao*abc and Xingdong Zhangf
aGuangxi Engineering Center in Biomedical Materials for Tissue and Organ Regeneration, Guangxi Collaborative Innovation Center for Biomedicine, The First Affiliated Hospital of Guangxi Medical University, 530021, Nanning, China. E-mail:;; Fax: +86-07715350975; Fax: +86-07715350189; Tel: +86-07715358132 Tel: +86-07715350189
bDepartment of Orthopaedics Trauma and Hand Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, China
cGuangxi Key Laboratory of Regenerative Medicine, The First Affiliated Hospital of Guangxi Medical University, 530021, Nanning, China
dCenter for Animal Experiment, The First Affiliated Hospital of Guangxi Medical University, 530021, Nanning, China
eDepartment of Cell Biology & Genetics, School of Premedical Sciences, Guangxi Medical University, 530021, Nanning, China
fNational Engineering Research Center for Biomaterials, Sichuan University, 610064, Chengdu, China

Received 2nd January 2018 , Accepted 3rd March 2018

First published on 6th March 2018

In this study, we report that the intra-hydrogel culture system mitigates the transformation of mesenchymal stem cells (MSCs) induced by two-dimensional (2D) expansion. MSCs expanded in monolayer culture prior to encapsulation in collagen hydrogels (group eMSCs-CH) featured impaired stemness in chondrogenesis, comparing with the freshly isolated bone marrow mononuclear cells seeded directly in collagen hydrogels (group fMSCs-CH). The molecular mechanism of the in vitro expansion-triggered damage to MSCs was detected through genome-wide microarray analysis. Results indicated that pathways such as proteoglycans in cancer and pathways in cancer expansion were highly enriched in eMSCs-CH. And multiple up-regulated oncoma-associated genes were verified in eMSCs-CH compared with fMSCs-CH, indicating that expansion in vitro triggered cellular transformation was associated with signaling pathways related to tumorigenicity. Besides, focal adhesion (FA) and mitogen-activated protein kinase (MAPK) signaling pathways were also involved in in vitro expansion, indicating restructuring of the cell architecture. Thus, monolayer expansion in vitro may contribute to vulnerability of MSCs through the regulation of FA and MAPK. This study indicates that intra-hydrogel culture can mitigate the monolayer expansion induced transformation of MSCs and maintain the uniformity of the stem cells, which is a viable in vitro culture system for stem cell therapy.

1. Introduction

Mesenchymal stem cells (MSCs) have been widely used in tissue engineering due to their self-renewal and differentiation capacity, as well as low immunogenicity, multipotency, etc.1 For stem cell based therapy, MSCs usually undergo the process of expansion in vitro in order to obtain sufficient cells. However, in vitro culture has adverse effects on the stemness of MSCs, which greatly influence the cell phenotype, survival rate, and migration.2 In our previous study, we found that compared with freshly extracted MSCs, early passaged MSCs presented impaired stemness and pluripotency with discounted therapeutic effects in cartilage repair.3 Our findings suggested that in vitro culture and expansion are not recommended for cell-based therapy. However, the underlying mechanism of expansion induced transformation of MSCs is still to be uncovered.

It is generally accepted that three-dimensional (3D) culture is preferred over monolayer systems and has a critical impact on physiological cell–cell and cell–extracellular matrix (ECM) interactions, which facilitates the process of phenotypic maintenance and functional differentiation of cells.4 Among the various 3D culture systems, hydrogels are highly recommended because they are hydrophilic cross-linked networks which provide a simulating microenvironment with meticulous manipulation over biophysical and biochemical signaling stimuli, such as nutrient release, oxygen supply, cellular morphological and mechanical maintenance.5,6 Most importantly, intra-hydrogel culture may help preserve the cellular behavior in a natural state, mimicking the in vivo tissue microenvironment.7,8 Collagen hydrogels have favorable biocompatibility, biodegradability and cell-adhesive properties. They are “golden standard” for the repair of osteochondral defects9 because they are highly hydrated three-dimensional networks mimicking the biochemical complexity of articular cartilage, which may be favorable to preserve cells in their natural state.10 Thus, collagen hydrogels may provide biomimetic environment to reduce the impact of flat-surface on MSCs as much as possible and may be a useful tool to compare freshly isolated MSCs (fMSCs) and monolayer cultured MSCs (eMSCs) to study the impact of monolayer culture on MSCs.

In this study, we used an intra-hydrogel culture system based on collagen hydrogels to compare fMSCs with eMSCs and to investigate the mechanism of two-dimensional (2D) culture induced transformation of MSCs. Both rabbit and human derived MSCs were used. This study may provide reference for the study of cell therapy by using expanded cells.

2. Materials and methods

2.1 Cell isolation and culture

Research on human subjects was designed following the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects11 and was approved by the Review Board of the First Affiliated Hospital of Guangxi Medical University (Nanning, China). Bone marrow aspirates were harvested from five healthy hematopoietic stem cell donors, after obtaining written informed consent. Twenty milliliters of bone marrow from each donor were assigned to MSC generation; heparin was added as an anticoagulant. Human bone marrow mesenchymal stem cells (MSCs) were obtained by bone marrow puncture using Ficoll-Paque PREMIUM (GE Healthcare Life Sciences, USA). Rabbit freshly isolated bone marrow MSCs (fMSCs) were harvested using a bone marrow mononuclear cell isolation kit (TBD2013CRA, Tian Jin Hao Yang Biological Manufacture Co. Ltd, China) as recommended by the manufacturer. Some of the isolated cells were cultured in 100 mm-diameter tissue culture dishes (Corning, USA) in culture media containing alpha-modified Eagle's medium (αMEM, Gibco, USA), 1% penicillin–streptomycin (Solarbio, Beijing, China) and 10% fetal bovine serum (FBS, Zhejiang Tianhang Biotechnology Ltd, China), at 37 °C in a 5% CO2 humidified atmosphere. The medium was changed every 3 days. The cells from human and rabbit were used in this study. The third passage MSCs were termed as eMSCs.

2.2 Phenotypes of MSCs

Phenotypes of human fMSCs and eMSCs obtained from 5 donors were studied by flow cytometry. Briefly, 1 × 106 cells were incubated with primary antibodies for two hours at 4 °C and then treated with the secondary antibodies for 30 minutes at 4 °C. CD73, CD90, CD105 and the isotype control antibodies were purchased from Abcam (Shanghai, China). The secondary antibody was GoatAnti-Rabbit IgG H&L (Alexa Fluor® 488, Abcam, Shanghai, China). Flow cytometric experiments were performed with a FACScan flow cytometer (Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA).

2.3 Apoptosis assay of MSCs

Apoptosis was performed using an Apoptosis Kit (Solarbio, Beijing, China) according to the manufacturer's instructions. Briefly, human fMSCs and eMSCs (with the same cell number of 105 cells per sample) were harvested and then incubated with AnnexinV-FITC and propidium iodide (PI) at room temperature in the dark for about 10 min. After that, 500 μL PBS was added to the cells, then vortexed gently and subjected to flow cytometry.

2.4 Intra-hydrogel culture of MSCs

Calf skin-derived type I collagen was prepared as previously described.9,10 A 15 mg mL−1 solution was neutralized with 1 M NaOH and stored at 4 °C before use. Human MSCs were first harvested and then mixed with neutralized collagen solution by vortex at a concentration of 107 cells per ml. Cell-collagen composites (fMSCs-CH and eMSCs-CH) were gelated after 10 min in 37 °C, and then cultured in culture media.

2.5 Proliferation assay of MSCs and MSCs-CH

Proliferation assay was conducted at day 7, 14, and 21 using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide). Briefly, for fMSCs-CH and eMSCs-CH, cell-gel composites (106 cells in 100 μL gel) were incubated in culture medium with MTT (5 mg mL−1) (culture medium[thin space (1/6-em)]:[thin space (1/6-em)]MTT = 5[thin space (1/6-em)]:[thin space (1/6-em)]1) for 4 h at 37 °C. After the supernatants were removed, the cell-gel composites were cool pulverized using a tissuelyser (Shanghai Jinxin Industrial Development Co. Ltd, Shanghai, China). For 2D cultured samples, 106 cells were seeded on the dishes. 2D cultured cells were incubated in culture medium with MTT (5 mg mL−1) (culture medium[thin space (1/6-em)]:[thin space (1/6-em)]MTT = 5[thin space (1/6-em)]:[thin space (1/6-em)]1) for 4 h at 37 °C. For each sample, MTT formazan crystals were obtained after 1 mL of DMSO was added. Every 200 μL of MTT formazan crystals was transferred to a well (96-well plate) and read using a microplate reader (Thermo, Shanghai, China) at 490 nm.

2.6 Chondrogenesis of intra-hydrogel cultured MSCs

Human fMSCs-CH and eMSCs-CH were cultured in chondrogenic defined medium containing α-MEM (Gibico, USA), 10% FBS, 10 ng ml−1 TGF-β1 (Propetech, USA), 100 nM dexamethasone (Sigma, USA), 1% insulin–transferrin–selenium solution (Gibico, USA), and 50 μg ml−1 ascorbic acid (Sigma, USA). The medium was replaced every 3 days.

2.7 Microarray analysis and data processing

Rabbit MSCs and MSCs-CH were collected for microarray analysis. Briefly, eMSCs and fMSCs at day 0 and after intra-cultured in collagen for 21 days were obtained. Microarray analysis was performed by Shanghai KangChen Bio-tech (China) on the Agilent Array platform in three replicates. The subsequent steps were performed according to the Agilent Whole Genome Oligo Microarray (one-color) protocol.

Data preprocessing and normalization were performed using GeneSpring GX v12.1. For the analysis, two factors were used: passage (eMSCs and fMSCs) and hydrogel (plate and intra-hydrogel). Gene Ontology (GO) term analysis was performed using DAVID ( Altered gene expression was compared with the expression of all genes on the array, and over-represented GO terms were identified automatically by the software with p < 0.05 considered significant.13 Overrepresentation of genes in a KEGG pathway was present if a larger portion of genes within that pathway were differentially expressed.13 And gene interaction analysis was evaluated using GENE MANIA ( To select differentially expressed genes, ratio change threshold values of g 1.0 or e 0.01 were used. Hierarchical clustering was performed using log 2-transformed data in Cluster 3.0, and heat maps were generated by the Treeview program.

2.8 Real-time PCR

Real-time PCR was performed to validate the microarray data; we examined the expansion-induced expression of several genes by comparing MSCs and MSCs-CH (both in human and rabbit). These genes include caspase 3 (CASP3) (human forward: 5′-GGGTTAACCGAAAGGTGGCA-3′, reverse: 5′-CTGCAGCATGAGAGTAGGTCA-3′; rabbit forward: 5′-GTGGCATCGAGACAGACAGT-3′, reverse: 5′-CCTCCTCCGAATTTCGCCA-3′), jun proto-oncogene (JUN) (human forward: 5′-GGAGACAAGTGGCAGAGTCC-3′, reverse: 5′-CCAAGTTCAACAACCGGTGC-3′; rabbit forward: 5′-CAAGTGCCGGAAAAGGAAGC-3′, reverse: 5′-CTGCGTTAGCATGAGTTGGC-3′), protein kinase C beta (PRKCB) (human forward: 5′-ATGGCAACAGAGACCGGATG-3′, reverse: 5′-CCATAGTGCACTCCACGTCA-3′; rabbit forward: 5′-GTGGCTTACCCCAAGTCCAT-3′, reverse: 5′-CTGGTCGGGAGGTGTTAGGA-3′), matrix metallopeptidase 2 (MMP2) (human forward: 5′-ACCAGCTGGCCTAGTGATGA-3′, reverse: 5′-CCGCATGGTCTCGATGGTAT-3′; rabbit forward: 5′-GAAGGCCGTGTTCTTTGCAG-3′, reverse: 5′-GTCTACTCGCTGGACATCGG-3′), v-myc avian myelocytomatosis viral oncogene homolog (MYC) (human forward: 5′- ATCACAGCCCTCACTCAC-3′, reverse: 5′-ACAGATTCCACAAGGTGC-3′; rabbit forward: 5′-CTCCATGAAGAGACACCGCC-3′, reverse: 5′-ACCCTGACCTTTTGGTGGAG-3′), signal transducer and activator of transcription 3 (STAT3) (human forward: 5′-AACCTGGCCTTTGGTGTTGA-3′, reverse: 5′-AGGTTGTAAGCACCCTCTGC-3′; rabbit forward: 5′-ATCCTGGTGTCTCCACTCGT-3′, reverse: 5′-CACTACCTGGGTCAGCTTCAG-3′), protein kinase C gamma (PRKCG) (human forward: 5′-ATCCACGTAACTGTTGGCGA-3′, reverse: 5′-TTCACCGTTCGGGTCTTCTG-3′; rabbit forward: 5′-GTGGACGGGTGGTACAAGTT-3′, reverse: 5′-CCCGCTCATACAACTCCAGG-3′), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB) (human forward: 5′-GCCTTTGGAACTTGGCACTG-3′, reverse: 5′-AGAGCTCCAAAGCAGCAGAG-3′; rabbit forward: 5′-AAGACGCCCTTCTGAACTGG-3′, reverse: 5′-TGTGTCTGTCACCAATCCCG-3′), and v-raf-1 murine leukemia viral oncogene homolog 1 (RAF1) (human forward: 5′-CTGGCTCCCTCAGGTTTAAGA-3′, reverse: 5′-AAGGCAGTCATGCAAGCTCA-3′; rabbit forward: 5′-AGTGCTGTGCTGTGTTCAGA-3′, reverse: 5′-GGCAAGCTTCAGGAACGTCT-3′). Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, USA) according to the manufacturer's protocol. The subsequent steps were performed according to the manufacturer's instructions. All primers were designed based on established GenBank sequences, and the amplification of GAPDH (human forward: 5′-CTATAAATTGAGCCCGCAGC-3′, reverse: 5′-GACCAAATCCGTTGACTCCG-3′; rabbit forward: 5′-GTCATCATCTCAGCCCCCTC-3′, reverse: 5′-GGATGCGTTGCTGACAATCT-3′) was used as a control to assess PCR efficiency. The comparative Ct method was used for gene expression quantification.

2.9 Western blotting

Total proteins from samples were extracted using RIPA Lysis Buffer (Beyotime Biotechnology, Beijing, China) and 1 mM PMSF (Beyotime Biotechnology, Beijing, China). Western blotting was performed using standard protocols. Collagen type 2 (COL2A1), sex determining region Y-box 9 (SOX9), CASP3, JUN and GAPDH were purchased from Boster Biological Technology (Wu Han, China). Samples were all revealed with Alexa infrared dye-conjugated secondary antibodies (Goat Anti-Rabbit IgG Alexa Fluor® 790, Invitrogen, USA), and then scanned via an Odyssey Infrared Imaging System (LI-COR). The relative expression of the proteins was quantified using ImageJ2x.

2.10 Immunostaining and analysis

Immunostaining was performed according to the manual introduction of VECTASTAIN® Elite® ABC Kit (Vector Laboratories, America). COL2A1, MYC and JUN antibodies were obtained from Boster Biological Technology (Wuhan, China). The analysis was performed with an upright microscope (Olympus BX53, Japan).

2.11 Xenograft of MSCs and MSCs-CH in vivo

All animal studies were approved by the Animal Care and Welfare Committee of Guangxi Medical University (Nanning, China). Immunodeficient mice of 4–5 weeks of age were anesthetized with a solution of ketamine and xylazine prior to the subcutaneous implantation of MSCs into the back. In order to facilitate implant establishment, MSCs (1 × 107) from both human and rabbits (eMSCs and fMSCs) were mixed with 100 μL normal saline/collagen for each sample prior to implantation. Perpendicular diameters of injected cells were measured with calipers every week for 8 weeks.

2.12 Statistical analysis

Statistical analysis was evaluated by Student's t-test using SPSS 16.0 (IBM, USA). Data were obtained using GraphPad Prism 5.0 (GraphPad Software). Data were represented as means ± SD and p-value < 0.05 was consider significant.

3. Results

3.1 Vulnerability of expanded MSCs

By using flow cytometry, we analyzed the surface markers known to be associated with human MSCs. Both fMSCs and eMSCs were positive for CD73, CD90 and CD105 (Fig. 1A and B). Intriguingly, even though the positive rate of eMSCs was lower than fMSCs, no significant differences were observed between them.
image file: c8bm00007g-f1.tif
Fig. 1 In vitro vulnerability of intra-hydrogel cultured human MSCs that experienced monolayer expansion. Representative phenotype flow cytometry of human (a) fMSCs and (b) eMSCs, n = 5, mean ± SD. Representative Annexin-V/PI flow cytometry of (c) fMSCs and (d) eMSCs; the apoptosis percentage was set as: Annexin-V + PI-cells, n = 5, mean ± SD. COL2A1 immunofluorescence of human fMSCs. (e) Proliferation rate of fMSCs, eMSCs, fMSCs-CH and eMSCs-CH, fMSCs; * indicates the difference between human fMSCs and eMSCs at the same day and # indicates the difference between 2D expansion and intra-hydrogel within the same human MSCs. (f) fMSCs-CH and (g) eMSCs-CH after chondrogenic induction for 21 days. Scale bar: 100 μm. (h) Representative blots and their fold induction of COL2A1 and SOX9 after chondrogenic induction for 7, 17 and 21 days. n = 3. Error bars, mean ± SD.*, # indicates p < 0.05.

Apoptosis of fMSCs and eMSCs was measured by analyzing phosphatidylserine using Annexin-V/PI. As shown in Fig. 1C, nearly 91% of fMSCs were viable (Annexin-VPI), whereas 7% of fMSCs were apoptotic (Annexin-V+PI), and the mean percentage of fMSCs undergoing apoptosis was 6.84 ± 0.05%. Compared with this, the eMSCs viability shown in Fig. 1D was 77%, with an apoptosis of 19%, and the mean percentage of eMSCs undergoing apoptosis was 18.09 ± 0.07%.

MTT assay was performed to evaluate the proliferation of fMSCs and eMSCs both in monolayer and intra-hydrogel culture. As shown in Fig. 1E, the difference between fMSCs and eMSCs in both 2D and 3D culture systems is minor at day 7, 14 and 21. In contrast to monolayer cultured MSCs (fMSCs and eMSCs) which showed minimal proliferation, intra-hydrogel MSCs (fMSCs-CH and eMSCs-CH) proliferated increasingly over time. At each time point, fMSCs proliferated more than eMSCs in the same culture system (p < 0.05).

3.2 Chondrogenesis of MSCs-CH

To evaluate the chondrogenic potential of human eMSCs and fMSCs, eMSCs and fMSCs were cultured with collagen and chondrogenic media for 7, 14 and 21 days. Immunohistochemistry staining of COL2A1 was measured after 21 days of chondrogenic induction. As shown in Fig. 1F and G, there were more chondrogenic specific markers detected in the fMSCs group than in the eMSCs group. Chondrogenic marker proteins were detected by western blotting (Fig. 1H). The expression levels of COL2A1 and SOX9 were higher in the fMSCs-CH group than in the eMSC-CH group (p < 0.05). In addition, with the increase of chondrogenic induction time, both COL2A1 and SOX9 increased, which indicated a success model of in vitro chondrogenic induction.

3.3 Comparative profiling of MSCs and MSCs-CH

In order to investigate the effects of intra-hydrogel culture on MSCs, we compared rabbit eMSCs, fMSCs, eMSCs-CH and fMSCs-CH by microarray analysis. Scatter-plot (Fig. 2A) and Volcano plot (Fig. 2B) were used to evaluate the variation between eMSCs and fMSCs cultures on plate and intra-hydrogels. As we can see, the differences between eMSCs and fMSCs in 2D expansion were obvious, while less differences showed between fMSCs-CH and eMSCs-CH.
image file: c8bm00007g-f2.tif
Fig. 2 (a) Scatter-plot, (b) Volcano plot and (c) pathway enrichment analysis of rabbit fMSCs and eMSCs before and after intra-hydrogel culture in 3D collagen.

The temporal patterns of activated genes associated with MSC expansion are presented in Fig. 2C. Genes involved in focal adhesion (FA), pathways in cancer (PC), proteoglycans in cancer (PGC), and the MAPK signaling pathway are significantly differentially expressed between eMSCs and fMSCs, which were associated with MSC expansion. However, these pathways have no effect when MSCs were cultured with collagen.

The differentially expressed genes in signaling pathways associated with expansion are listed in Table 1. A total of 54 genes affected by cell expansion in vitro were involved in FA, 89 genes were involved in PC, 58 genes were involved in PGC and 53 genes were involved in the MAPK signaling pathway. The interaction network of genes in these four vital pathways is presented in Fig. 3. The corresponding molecules in these pathways were investigated by hierarchical clustering analysis (Fig. 4). The results indicated that these molecules might contribute to changes in MSC tumorigenesis.

image file: c8bm00007g-f3.tif
Fig. 3 Interaction of genes in (a) proteoglycans in cancer signaling pathway, (b) focal adhesion signaling pathway, (c) pathways in cancer and (d) MAPK signaling pathway.

image file: c8bm00007g-f4.tif
Fig. 4 Heat map showing the molecules in the pathways performed by hierarchical clustering analysis.
Table 1 KEGG signaling pathways associated with MSCs transformation
Pathway title Count Genes down-regulated Genes up-regulated
Threshold: p-value < 0.05.

3.4 Intra-hydrogel culture mitigates the 2D expansion induced transformation of MSCs

To confirm the reliability of the microarray data, we examined the expansion-induced expression of several critical factors by western blotting analysis (Fig. 5A and 6A). CASP3 and the cleaved CASP3 showed reduction in eMSCs, while the JUN level was increased in eMSCs. Immunofluorescence of fMSCs and eMSCs (both rabbit (Fig. 5B) and human (Fig. 6B)) at day 0 was also the result of western blot. Expression of JUN was conjugated with FITC (green) and MYC was conjugated with CY5 (red) in immunostaining. As shown in Fig. 5B and 6B, our immunostaining of JUN and MYC in the fMSC group was lower than that in eMSCs at day 0. In addition, our PCR showed that CASP3, MMP2 and STAT3 expression was significantly down-regulated after MSC expansion in vitro (in both rabbit (Fig. 5C) and human (Fig. 6C)). By contrast, PRKCB, PRKCG, MYC, JUN, PIK3CB and RAF1 were increased in eMSCs. However, after intra-hydrogel culture, JUN and STAT3 were up-regulated in eMSC-CH, which were higher than in fMSC-CH. However, PRKCB, PRKCG and PIK3CB cannot be detected in the human data, which may indicate some difference of MSCs between human and rabbit. Expansion in vitro accelerates MSCs spontaneously malignant transformation through proteoglycans in cancer, the focal adhesion signaling pathway, MAPK signaling pathway and pathways in cancer. And MSCs cultured in collagen showed great uniformity.
image file: c8bm00007g-f5.tif
Fig. 5 (a) Representative blots and fold induction of CASP3, cleaved CASP3 and JUN in rabbit fMSCs and eMSCs. n = 3. Error bars, mean ± SD.* indicates p < 0.05. (b) Immunofluorescence of JUN and MYC in rabbit fMSCs and eMSCs. Scale bar: 50 μm. (c) CASP3, JUN, MMP2, STAT3, PRKCB, PRKCG, MYC, PIK3CB and RAF1 mRNA expression of rabbit fMSCs and eMSCs cultured on plate and intra-hydrogels. n = 3. Error bars, mean ± SD.* indicates the difference between rabbit fMSCs and eMSCs at the same day and # indicates the difference between 2D expansion and intra-hydrogel within the same rabbit MSCs. * #, p < 0.05.

image file: c8bm00007g-f6.tif
Fig. 6 (a) Representative blots and fold induction of CASP3, cleaved CASP3 and JUN in human fMSCs and eMSCs. n = 3. Error bars, mean ± SD.* indicates p < 0.05. (b) Immunofluorescence of JUN and MYC in human fMSCs and eMSCs. Scale bar: 50 μm.(c) CASP3, JUN, MMP2, MYC, STAT3 and raf1 mRNA expression of human fMSCs and eMSCs cultured on the plate and intra-hydrogels. n = 3. Error bars, mean ± SD.* indicates the difference between human fMSCs and eMSCs at the same day and # indicates the difference between 2D expansion and intra-hydrogel culture within the same human MSCs. * #, p < 0.05.

3.5 Xenograft tumorigenity

To confirm the role of 2D expansion in MSC maldifferentiation, fMSCs and eMSCs from human and rabbits were implanted subcutaneously in immunodeficient mice (n = 5). However, no signs of illness or tumor formation were detected in mice injected with eMSCs and fMSCs from human or rabbits. And the perpendicular diameters of injected eMSCs and fMSCs stayed the same up to 4 weeks and then lessened slightly up to 8 weeks with no significant differences. However, the volume of eMSCs-CH and fMSCs-CH showed no differences up to 8 weeks.

4. Discussion

Our previous study verifies that compared with fresh MSCs, in vitro expanded MSCs exhibit inferior bioactivity and impaired therapeutic effects in cartilage repair.3 In this study based on sequencing analysis, the highly enriched proteoglycans in cancer/pathways in cancer and multiple up-regulated oncoma-associated genes were found in eMSCs, indicating that 2D expansion may contribute to MSC transformation in vitro. Furthermore, intra-hydrogel culture based on collagen could mitigate the 2D expansion induced transformation of MSCs.

In both rabbit and human originated eMSCs representative of short-term cultured stem cells, decreased chondrogenic potential was evidenced, as compared with freshly isolated fMSCs. Telomerase and chromosome abnormalities were also found in eMSCs.3 These findings indicated that in vitro culture expanded stem cells are not safe, even for short-term. Since in vitro environment greatly differs from the human body, it can hardly provide sufficient “natural” cells for regenerative medicine.3 The safety of passaged cells should be suspected.

Although the surface markers (CD73, CD90 and CD105) of human fMSCs and eMSCs presented no significant differences, there are more apoptotic cells in human eMSCs than in fMSCs. This suggested that 2D expansion may contribute to vulnerability of MSCs in vitro. Notably, MSCs did not proliferate in monolayer culture after 7, 14 and 21 days because they became too crowded after a long period of culture, which inhibited their proliferation. Although fMSCs are heterogeneous in cell population, interindividual differences are not well notable as eMSCs, which can also be maintained in collagen hydrogels. Comparative profiling of fMSCs with eMSCs provides unique insight into the changes that stem cells undergo during expansion. Important genes cross-linked with pathways were analyzed via clustering in the heatmap (Fig. 4, up-regulated genes in red and down-regulated genes in green). The differences among the fMSC groups were minor. However, notable deviations were observed among these MSCs, indicating that in vitro expansion greatly increased the heterogeneity of the stem cells. A minor change in in vitro culture conditions may elicit a cascade of modifications in cell behavior. Moreover, the 3D collagen gel is more realistic than monolayer culture for in vitro culture because it can maintain the uniformity of the cells, as indicated by the limited differences between the intro-hydrogel fMSC-CH and eMSC-CH groups. In addition, 3D culture ensured the constant proliferation of stem cells.

It has been reported that long-term passaging may lead to malignant transformation of adult stem cells.14–18 However, tumorigenesis of MSCs originating from short-period expansion has seldom been reported. In our pathway enrichment analysis, proteoglycans in cancer (PGC) and pathways in cancer (PC) were highly enriched pathways in eMSCs, indicating that the flattened MSCs were not safe. Most of the crucial genes verified by real-time PCR were oncoma-associated genes, such as PIK3CB,19 RAF1,20 CASP3,21 STAT3,22 MYC23,24 and JUN.25 PIK3CB19 and RAF120 represent important therapeutic targets for tumors.

The in-depth analysis indicated the involvement of focal adhesion (FA) and MAPK in 2D expansion. FA is quantitatively related to the strength of the cell.26 FA proteins may control the remodeling of actin cytoskeleton networks and further control MSC commitment.27–29 The PIK3CB and STAT3 genes are not only related to oncoma but also related to the cellular microarchitecture. The expansion induced by FA indicates that the monolayer-cultured MSCs underwent restructuring of the cell architecture.30 We speculate that genetic alterations of cells occur following the sensing of a flat surface; in other words, expansion may proceed to tumorigenicity through the regulation of FA. Another important pathway involved in the expansion of stem cells, the MAPK pathway, has been reported to regulate the self-renewal process and differentiation commitment of embryonic stem cells.31,32 This pathway also plays an important role in skewed expanded MSC differentiation.33,34

Some genes were also related to pluripotency of MSCs. In addition to acting as an oncoma gene, STAT3 is also involved in stem cell transplant-associated diseases.35 During the self-renewal and lineage commitment processes, PRKC isoforms are repressed in mouse embryonic stem cells.36 The up-regulation of PRKCB and PRKCG induced by expansion may contribute to the tumor transformation of MSCs. JUN activates mesenchymal-related genes, and its suppression benefits pluripotency.25 The increase in JUN in the eMSC group indicated suppression of mesenchymal properties and stemness. In the meantime, MYC overexpression kills normal cells and provokes cancer,23,37 and the high level of MYC in eMSCs suggest its potential effects in tumor formation of MSCs. However, no tumor formation showed in the xenograft study. And as is known, MYC is also one of the transcript factors usually used in reprograming normal cells into induced pluripotent stem cells (iPSCs), indicating the important role in stem cell multipotency.38,39 PGC and PC pathways were activated by 2D expansion, as revealed by upregulated MYC, which may be due to a self-renewal mechanism for reversing the apoptosis of MSCs. Conversely, fMSCs-CH or eMSCs-CH exhibited much less enrichment of the PGC and PC pathways, which also corroborates the finding that 2D expansion may be one of the important cues responsible for MSCs vulnerability. This study reflects the findings that cells growing on a flat surface are forced to adopt unnatural characteristics, such as an aberrant flattened morphology, which may hamper stem cell proliferation40 and change the biological properties, functions and even genetic characteristics of the cells.15,41–43

In conclusion, this study demonstrated that MSCs were more vulnerable in vitro by 2D expansion, even though the culture time is short-term, which were highly associated with signaling pathways related to tumorigenicity, focal adhesion and MAPK. Also, intra-hydrogel culture based on collagen mitigates the 2D expansion induced transformation of mesenchymal stem cells during chondrogenesis.

Conflicts of interest

The authors confirm that this article content has no competing interest.

Animal experimental statement

Conditions of the housing facilities, including a barrier housing facility and an ordinary housing facility, keep-up with national standard “Laboratory Animal—Requirements of Environment and Housing Facilities” (GB 14925-2010) of China. The care of laboratory animals and the animal experimental operation are conforming to “Administration Rule of Laboratory Animal”, etc. of China.


This work has been financially supported by National key research and development program of China (2016YFB0700804), National Natural Science Fund of China (Grant No. 81760326), Guangxi Scientific Research and Technological Development Foundation (GuikeAB16450003), High level innovation teams and outstanding scholars in Guangxi Universities (The third batch), Distinguished Young Scholars Program of Guangxi Medical University, and Innovation Project of Guangxi Graduate Education (YCSZ2015108).


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These authors contributed equally to this work.

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