Characterization of human telomerase reverse transcriptase immortalized anterior cruciate ligament cell lines

Patrick Prager; Matthias Schieker; Franz Jakob; Denitsa Docheva; Christian Konrads; Andre Steinert

doi:10.1016/j.bj.2019.05.005

. 2019 Dec 23;42(6):371–380. doi: 10.1016/j.bj.2019.05.005

Characterization of human telomerase reverse transcriptase immortalized anterior cruciate ligament cell lines

Patrick Prager ^a, Matthias Schieker ^b, Franz Jakob ^a, Denitsa Docheva ^c, Christian Konrads ^a,^d,^∗,¹, Andre Steinert ^a,^e,¹

PMCID: PMC6962762 PMID: 31948601

Abstract

Background

The anterior-cruciate-ligament (ACL) contains mesenchymal stem cells (ACL-MSCs), suggesting the feasibility of regenerative treatments of this tissue. The immortalization of isolated cells results in cell-lines applicable to develop cell-based therapies. Immortal cell lines eliminate the need for frequent cell isolation from donor tissues. The objective of this study was to characterize cell lines that were generated from isolated ACL-MSCs using TERT gene transfer.

Methods

We isolated ACL-MSCs from human ACLs derived at the time of ACL reconstruction surgery or total knee arthroplasty. We generated cell lines and compared them to non-immortalized ACL-MSCs. We assessed the cellular morphology and we detected surface antigen expression. The resistance to senescence was inferred using the beta galactosidase activity. Histology, immunohistochemistry, and reverse transcriptase polymerase chain reaction (RT-PCR) were used to evaluate the multilineage differentiation capacity.

Results

The morphology of hTERT-ACL-MSCs was similar to ACL up to the last assessment at passage eight. We detected a strong surface expression of CD44, CD90, CD105, and STRO-1 in hTERT-ACL-MSCs. No substantial reduction in the ATP activity was observed in hTERT-ACL-MSCs.

Conclusion

Cell lines generated from ACL-MSCs maintain their morphology, surface antigen expression profile, and proliferative capacity; while markers of senescence appear to be reduced. These cell-lines maintained their multilineage differentiation capacity. The demonstrated model systems can be used for further development of new cell-based regenerative approaches in anterior cruciate ligament research, which may lead to new therapeutic strategies in the future.

Keywords: Anterior cruciate ligament, Mesenchymal stem cells, Immortalization, Senescence, Multilineage differentiation

At a glance of commentary

Scientific background on the subject

The anterior cruciate ligament (ACL) contains mesenchymal stem cells. Immortalization of those cells results in cell-lines, which might be used for regenerative therapies in the future. The objective of this study was to characterize these ACL-derived cell-lines.

What this study adds to the field

The study demonstrates that cell-lines generated from ACL-derived stem cells maintain their morphology, surface antigen expression profile, and proliferative capacity while markers of senscence are reduced. The developed cell-lines maintained their multilineage differentiation capacity. The demonstrated model can be used for further development of new cell-based regenerative approches in ACL research.

Injuries to the anterior cruciate ligament (ACL) result in a gross destabilization of the joint and the development of osteoarthritis. The functional restoration of the ligament is a key component in the prevention of secondary meniscal tears [1], [2]. The current standard of care is the surgical replacement of the torn ACL with an autologous semitendinosus or patellar tendon graft [3], [4]. However, high-level evidence suggests that the current standard of care is not resulting in substantially improved subjective outcomes [5], [6]. Further radiographic signs of osteoarthritis are observed in a high frequency after ACL injury and reconstruction [7]. In order to improve outcomes after ACL injury, novel regenerative therapies are being explored, that might allow restoring the proprioceptive nerve functions of the native ACL and omitting the harvest of an autologous tendon graft.

Multipotent mesenchymal stem cells (MSCs) might be used for the development of cell-based regenerative therapies. They can be isolated from a variety of different tissues including bone marrow, adipose tissue, trabecular bone, and tendon [8], [9], [10], [11]. We have recently shown that cells isolated from torn ACLs of young donors possess a surface antigen expression profile highly similar to those of bone-marrow-derived MSCs (BMSCs) and that they were able to undergo multilineage differentiation [12], which qualifies them as MSC types [13]. Thus, the use of ACL-derived mesenchymal stem cells (ACL-MSCs) for regenerative therapies of the injured ACL appears attractive. However, ACL-MSCs can only proliferate in vitro for a limited number of times. By reducing the tendency to senescence using immortalization techniques, cell lines can be created. Those cell lines can improve the design and development of regenerative therapies by limiting the need of cell-isolations from donor tissues and reducing the variability introduced by different donors.

In this study, we have used human TERT gene transfer (encoding Telomerase reverse transcriptase) to immortalize ACL-MSCs derived from one young and one old donor. We hypothesized that the created cell lines (hTERT-ACL-MSCs) maintain the cellular morphology, surface antigen expression profile, and proliferative capacity of naive non-immortalized ACL-MSCs. Further we assessed, whether ACL-MSC cell lines maintain their capacity for multilineage differentiation.

Material and methods

Patient characteristics and tissue collection

ACL tissue was obtained from one young (17 years) and one old (73 years) ACL donor at the time of ACL reconstruction or joint replacement surgery, respectively; according to the protocol approved by our university's Ethics Committee and after informed consent of the patients and parents in case of minors.

Isolation and culture of ACL-MSCs

ACL-MSCs were isolated using collagenase digestion [12]. Tissue specimens were cut into smaller pieces followed by 12.5 U/μl collagenase NB4 (Serva Electrophoresis GmbH, Heidelberg, Germany) in complete medium consisting of Dulbecco's Modified Eagle Medium DMEM/Ham's F12 (PAA), 10% fetal bovine serum (FBS) (PAA Laboratories GmbH, Coelbe, Germany), 1% penicillin/streptomycin (PAA) and 50 mg/ml l-ascorbic acid 2-phosphate (Sigma Aldrich GmbH, Taufkirchen, Germany) for 18 h. The released cells were separated from tissue debris using a cell strainer, pelleted at 1200 g for 5 min, resuspended in complete medium, and seeded in 75 cm² flasks. For all subsequent differentiation protocols, cells were cultivated in an incubator at 37 °C and 5% CO₂ in a humidified atmosphere with media changes every 2–3 days. Cells were passaged using 1% trypsin (PAA) for 5 min for detachment, an equal volume of complete medium to stop the reaction, centrifugation at 1200 g for 5 min to pellet cells, and resuspension in complete medium, after which the cells were seeded at a minimal density of 5 × 10³ per ml in cell culture flasks.

Immortilization of ACL-MSCs

The viral overexpression of TERT (encoding human telomerase reverse transcriptase; hTERT) was used to immortalize third passage ACL-MSCs. The gene transfer was performed using a lentiviral vector, as previously described [14], [15].

Cell proliferation assay

The quantification of adenosine 5′ triphosphate (ATP) was used to assess the cell proliferation rate. The sensitive luminometric Cell Titer Glo® assay system (Promega, Mannheim, Germany) was used according to the manufacturer's instructions. Briefly, first passage ACL-MSCs were seeded at 1000 cells per well in 96 well plates (Thermo Fischer Scientific Nunc, Langenselbold, Germany) and cultured in 100 μL complete DMEM/F-12 media per well for 13 days, with media changes every 2 days. At days 2, 4, 6, 8, 10, and 13 the luminescence of 10 wells per cell type and donor was measured for 0.1 s in a luminometer after an equal volume of Cell Titer-Glo® Reagent was added per well followed by 5 min incubation at room temperature.

β-Galactosidase staining

Senescent cells were identified as previously described using the histological visualization of senescence-associated endogenous β-galactosidase activity [16], [17]. Briefly, following two washing steps with PBS (phosphate buffer solution), cells were fixed for 5 min in 2% (v/v) formaldehyde: 0.2% (v/v) glutaraldehyde (1:1). Following an additional PBS wash, cells were incubated with freshly prepared β-galactosidase staining solution at 37 °C without extra addition of carbon dioxide for 16 h. Subsequently, the monolayer was washed twice with PBS for 5 min, rinsed with deionized water and analyzed microscopically.

Chondrogenic differentiation

Chondrogenic differentiation was performed in a pellet culture system, as previously described [18]. Third passage cells were detached from the plastic surface of culture flasks using trypsin digestion, counted and 2.5 × 10⁵ cells were transferred in 50 ml canonical tubes containing 500 μl of either chondrogenic or control media, followed by centrifugation at 1500 g. Pellets were cultured in DMEM High Glucose medium supplemented with 1% penicillin/streptomycin, 40 ng/ml l-proline, 50 ng/ml l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 100 ng/ml pyruvate, 100 nM dexamethasone, 1% ITS (all Sigma-Aldrich, Schnelldorf, Germany), and human TGF-β1 (R&D Systems) for 28 days with the caps slightly opened to enable gas exchange. Control pellets were cultured in the absence of TGF-β1. RT-PCR of the chondrocyte marker genes ACAN (encoding aggrecan core protein), BGN (encoding biglycan), COMP (encoding cartilage oligomeric matrix protein) FMOD (encoding fibromodulin), as well as COL2A1 (encoding collagen alpha-1(II) chain) were used to gauge the degree of chondrogenic differentiation. COL10A1 (encoding collagen alpha-1(X) chain) expression was used to determine the degree of chondrocyte hypertrophy. Further qualitative histological assessment of alcian blue histology and type II collagen immunohistochemistry was performed.

Osteogenic differentiation

Osteogenic differentiation was performed in a monolayer culture system as previously described with slight modifications [18]. Cells were cultured in DMEM High Glucose medium containing 10% FBS, 1% penicillin/streptomycin, 10 ng/ml bone morphogenetic protein-2 (kindly provided by Prof. Dr. Walter Sebald, Emeritus Chair of Physiological Chemistry, Faculty of Medicine, University of Wuerzburg, Germany), 10 mM β-glycerophosphate, 50 ng/ml l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, and 100 nM dexamethasone (all Sigma Aldrich GmbH) for 28 days. Control cells were cultured in DMEM High Glucose containing 10% FBS and 1% penicillin/streptomycin only. RT-PCR of the osteocyte-specific marker gene ALPL (encoding alkaline phosphatase, tissue-nonspecific isozyme) and the qualitative assessment of alkaline phosphatase and alizarin red positive areas were performed to gauge the degree of osteogenic differentiation.

Adipogenic differentiation

Adipogenic differentiation was performed in a monolayer culture system according to a modified protocol described before [18]. Cells were incubated with DMEM High Glucose containing 10% FBS, 1% penicillin/streptomycin, 1 μg/ml insulin, 100 μM indomethacin, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 μM dexamethasone (all Sigma Aldrich) for a total time of 21 days. Control cells were cultured in DMEM High Glucose containing 10% FBS and 1% penicillin/streptomycin only. RT-PCR of the adipocyte-specific marker genes LPL (encoding lipoprotein lipase) and PPARG (encoding peroxisome proliferator-activated receptor γ), as well as the qualitative assessment of oil red O positive areas were performed to gauge the degree of adipogenic differentiation.

RNA isolation and RT-PCR

RNA was isolated using the NucleoSpin© RNA II kit (Machery-Nagel GmbH, Dueren, Germany) according to the manufacturer's instructions. The pellets of chondrogenic cultures were previously homogenized in the first buffer of the respective kit using a pestle. 1 μg of isolated RNA was transcribed into cDNA using the BioScript™ kit (Bioline GmbH, Luckenwalde, Germany) with random hexamer primers (Life Technologies GmbH, Darmstadt, Germany) according to the manufacturer's protocols. Gene expression was analyzed using RT-PCR with reaction mixtures containing 3 μl 10x reaction buffer (shipped with the MangoTaq™ from Bioline), 1 μl 10 mM dNTP mixture (Bioline), 1 μl of each forward and reverse primer corresponding to a concentration of 5 μM (See Table 1 for primers) and 3 μl MangoTaq™ polymerase (5000 U/ml, Bioline) and 1 μl of cDNA in 21.9 μl distilled water. The PCR was started by an initial denaturation step at 94 °C for 3 min followed by cycles of denaturation at 94 °C for 45 s, primer annealing for 45 s with primer specific temperatures illustrated in Table 1, and an elongation step at 72 °C for 1 min. Subsequent agarose gel electrophoresis was performed using 1.5% agarose gels (Merck) containing 0.5 μg/l ethidium bromide (AppliChem GmbH, Darmstadt, Germany) and 0.5x TBE buffer at 0.9 V/cm of gel length. Gels were documented photographically using the Bio Profile software (LTF, Wasserburg, Germany) and analyzed densitometrically using the GelAnalyzer software (http://www.gelanalyzer.com). For all targeted gene expression analyses EEF1A1 (encoding Elongation factor 1-alpha) was used as housekeeping gene.

Table 1.

Primers and PCR conditions.

Gene	Forward and reverse primer	Product length	Annealing temperature	MgCl₂ Concentration	Accession ID (NCBI)
Adipogenic markers
LPL	F: 5′-CTGCAAATGAGACACTTTCTC-3' R: 5′-CATGTCCTGGCAGTCTCTGA-3′	276 bp	51 °C	1.0 g/l	NM_000237
PPARG	F: 5′-GCTGTTATGGGTGAAACTCTG-3' R: 5′-TAAGGTGGAGATGCAGGCTC-3′	350 bp	51 °C	1.0 g/l	NM_005037, NM_015869, NM_138711, NM_138712
Osteogenic marker
ALPL	F: 5′-TGGAGCTTCAGAAGCTCAACACCA-3' R: 5′-ATCTCGTTGTCTGAGTACCAGTCC-3′	454 bp	51 °C	1.0 g/l	NM_000478, NM_001127501, NM_001177520
Chondrogenic markers
ACAN	F: 5′-GCCTTGAGCAGTTCACCTTC-3' R: 5′-CTCTTCTACGGGGACAGCAG-3′	392 bp	54 °C	1.0 g/l	NM_001135, NM_013227
BGN	F: 5′-AGTAGAGGCAGCTGCAGCCAC-3' R: 5′-GTGTCTGGACAGGTGCGGCAGC-3′	424 bp	53 °C	1.0 g/l	NM_001711
COL2A1	F: 5′-GGTATTGCTGGCTTCAAAGG-3' R: 5′-AGGACGACCATCTTCACCAG-3′	387 bp	58 °C	1.0 g/l	NM_001844, NM_033150
COMP	F: 5′-CAGGACGACTTTGATGCAGA-3' R: 5′-AGGCTGGAGCTGTCCTGGTA-3′	312 bp	54 °C	1.0 g/l	NM_000095
FMOD	F: 5′-ACCAGTGATAAGGTGGGCAG-3' R: 5′-AAGTAGCTATCGGGGACGGT-3′	368 bp	54 °C	1.0 g/l	NM_002023
Hypertrophic marker
COL10A1	F: 5′-GGTATTGCTGGCTTCAAAGG-3' R: 5′-AGGACGACCATCTTCACCAG-3′	468 bp	54 °C	1.0 g/l	NM_000493
Housekeeping gene
EEF1A1	F: 5′-AGGTGATTATCCTGAACCATCC-3' R: 5′-AAAGGTGGATAGTCTGAGAAGC-3′	235 bp	54 °C	1.0 g/l	NM_001402

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Illustrated are the forward and reverse primers to amplify specific gene products along with their associated product lengths (l_Amplicon), the annealing temperature (T_Anneal), the concentrations of MgCl₂ used in the reaction mixture (c_MgCl), and the accession ID at the Gene database of the National Center for Biotechnology Information (NCBI).

Results

Cellular morphology and surface antigen expression

In monolayer culture, eighth passage hTERT-ACL-MSCs possessed a spindle-shaped morphology similar to non-passaged ACL-MSCs [Fig. 1]. A strong expression of the surface markers CD44, CD90, CD105, and STRO-1 was observed in hTERT-ACL-MSCs [Fig. 2].

Fig. 2 — Immunophenotyping of hTERT-immortalized and non-immortalized primary ACL cells from young and old donors. A strong expression of the surface markers CD44, CD90, CD105, and STRO-1 was observed for immortalized cell-lines and non-immortalized primary ACL-MSCs of young and old donors. Bars indicating 200 μm (primary cells) or 500 μm (cell lines).

Proliferative capacity and cellular senescence

The short-term ATP activity assessed at day 2, 4, 6, 8, 10, and 13 within a single passage demonstrated the same pattern in hTERT-ACL-MSCs and ACL-MSCs. An increase in activity was observed in all groups until day 8. ACL-MSCs plateaued thereafter, while hTERT-MSCs demonstrated a slight increase in ATP activity until day 10. No substantial difference was observed between hTERT-ACL-MSCs and ACL-MSCs at all time points [Fig. 3]. Following long-term cultivation, ACL-MSCs demonstrated a high staining intensity for beta–galactosidase activity, indicating cellular senescence. In comparison, the staining intensity in hTERT-ACL-MSCs was reduced [Fig. 4].

Fig. 3 — Short-term proliferative capacity of hTERT-ACL-MSCs and non-immortalized ACL-MSCs. No substantial difference in the proliferative capacity was observed between hTERT-ACL-MSCs (diamonds) and the corresponding non-immortalized ACL-MSCs (squares).

Fig. 4 — β-galactosidase activity of hTERT-ACL-MSCs and non-immortalized ACL-MSCs after long-term culture. The staining intensity was reduced in hTERT-ACL-MSCs (left panels) when compared to non-immortalized ACL-MSCs (right panels), indicating a stronger senescent phenotype of the non-immortalized ACL-MSCs.