Human articular chondrocytes with higher aldehyde dehydrogenase

Osteoarthritis and Cartilage 24 (2016) 873e882

Human articular chondrocytes with higher aldehyde dehydrogenase activity have stronger expression of COL2A1 and SOX9 A. Unguryte y *, E. Bernotiene y, E. Bagdonas y, S. Garberyte y, N. Porvaneckas z, C. Jorgensen x k y Department of Regenerative Medicine, State Research Institute Center of Innovative Medicine, Vilnius, Lithuania z Vilnius University, Medicine Faculty, Vilnius, Lithuania ^pital Saint-Eloi, Universit x INSERM U1183, IRMB Ho e Montpellier 1, Montpellier, France ^pital Lapeyronie, Montpellier, France k CHU Ho

a r t i c l e i n f o

s u m m a r y

Article history: Received 10 July 2015 Accepted 24 November 2015

Objective: To determine in human articular chondrocytes the activity of Aldehyde dehydrogenase (ALDH), which are reported as stem/progenitor cell marker in various adult tissues and evaluate gene expression of ALDH1A isoforms. Design: ALDH activity was evaluated by flow cytometry with Aldefluor™ assay in cells, isolated from human osteoarthritic (OA) cartilage. Its coexpression with surface markers was identified. Cells were sorted according to ALDH activity, and gene expression in sorted populations (ALDHþ and ALDH) was analyzed by RTq-PCR with Taqman® assay. Results: About 40% of freshly isolated chondrocytes demonstrated ALDH activity that remarkably declined during monolayer culture. Markers CD54 and CD55 were significantly stronger expressed, while CD47, CD140b, CD146 and CD166 were depleted in ALDH-expressing (ALDHpos) cells. Gene expression analysis revealed significantly higher expression of chondrocyte-specific genes COL2A1, SOX9 and SERPINA1 and lower expression of osteogenic markers RUNX2 and osteocalcin (BGLAP) in sorted ALDHþ fraction. COL1A1, ACAN, ALPL and stem cell markers NANOG, OCT4, SOX2 and ABCG2 did not differ remarkably between the populations. Genes of isoenzymes ALDH1A2, ALDH1A3 and ALDH2 were strongly expressed, while ALDH1A1 was weakly expressed in chondrocytes. Only ALDH1A2 and ALDH1A3 were significantly enriched in ALDHþ fraction. Conclusions: We identified ALDH activity with significantly stronger expression of CD54 and CD55 in human articular chondrocytes. Gene expression of isotypes ALDH1A2, ALDH1A3 and ALDH2 was identified. Coexpression of ALDH activity with chondrogenic markers suggests its association with collagen II producing chondrocyte phenotype. Isotypes ALDH1A2 and ALDH1A3 can be associated with the ALDH activity in these cells. © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Keywords: Chondrocytes Chondrocyte markers Aldehyde dehydrogenase (ALDH) Cartilage Progenitors

Introduction Articular cartilage has poor regeneration capacity, therefore, cartilage injuries often lead to osteoarthritis and finally result in

* Address correspondence and reprint requests to: A. Unguryte, Department of Regenerative Medicine, State Research Institute, Centre of Innovative Medicine, Santariskiu 5, 08406, Vilnius, Lithuania. Tel: 370-615-20253. E-mail addresses: [email protected], [email protected] (A. Unguryte), [email protected] (E. Bernotiene), edvardas.bagdonas@ gmail.com (E. Bagdonas), [email protected] (S. Garberyte), narunas. [email protected] (N. Porvaneckas), [email protected] (C. Jorgensen).

total joint destruction. The reason for this low repair potential is not completely clear yet. Characterization of cell populations specific for the articular cartilage phenotype may contribute to understanding how maintenance of cartilage is regulated. This is of critical importance for the development of cell-based therapy and cartilage tissue engineering. In 1999 Storms et al.1 characterized aldehyde dehydrogenasebright (ALDH)br population in human umbilical cord blood, which was enriched for hematopoietic progenitors. Employing this technique, stem or progenitor cells were characterized in human bone marrow and umbilical cord blood2e4, muscle5e7, heart8, murine neural tissue and neurospheres9, as well as other tissues.

http://dx.doi.org/10.1016/j.joca.2015.11.019 1063-4584/© 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

874

A. Unguryte et al. / Osteoarthritis and Cartilage 24 (2016) 873e882

ALDHs are the group of enzymes that catalyze irreversible NAD(P)þ dependant oxidation of various aldehydes by converting them to carboxylic acids, which protect cells from cytotoxic effects of aldehydes. Some of the carboxylic acids, generated by ALDHs, such as all-trans retinoic acid (ATRA), an active metabolite of vitamin A; betaine (osmolyte); neurotransmitter e g-aminobutyric acid (GABA) are very important in physiology of various cells and tissues (reviewed in10). ALDH genes are divided into families based on their amino acid identity and substrate specificity. Three members of ALDH1A subfamily, ALDH1A1, ALDH1A2 and ALDH1A3, also known as retinaldehyde dehydrogenases (RALDH1, RALDH2 and RALDH3, respectively), are involved in Vitamin A metabolism, as they irreversibly synthesize ATRA from retinaldehyde and are crucial in regulating retinoic acid signaling (both ATRA and 9-cisretinal)10. ATRA signaling plays a critical role in various cell functions: it regulates gene transcription, cell differentiation, other signaling events and post-translational modification of proteins11. Subtle regulation of this signaling relies on spatio-temporal control of ATRA synthesis and degradation due to the diverse physiological activity of ATRA in different organs, whereas altered levels of endogenous retinoids (generated from retinol within cells) were found in many diseases of the lung, kidney, and central nervous system, contributing to pathophysiology12. Each of these isozymes fulfills an important, yet specific function that cannot be replaced by the others (reviewed in13). Recent study showed that not only ALDH1A1 enzymatic activity could be detected and sorted by Aldefluor™ assay, as previously reported, but also ALDH1A2 and ALDH2 activities, which were also inhibited by DEAB14. Though ALDH2 is very similar to ALDH1 isoforms and sometimes is qualified as a member of this family, it is a mitochondrial enzyme, which is not associated with retinoid signaling, but rather involved in alcohol metabolism10. Activation of ALDH1 isoforms and their expression levels appeared to be regulated strictly in a tissue-specific way, suggesting that only tissue-specific approach to modify regulation of ALDH1As can result in therapeutic effects11. However, no analysis of ALDH activity, isotype composition and functions has so far been performed in adult cartilage. Aim of the present study was characterization of ALDHpositive populations and analysis of association between ALDH activity and the level of chondrocyte differentiation. We evaluated ALDH activity, gene expression and coexpression of cell surface markers with ALDH in chondrocytes, isolated from human OA articular cartilage samples. To the best of our knowledge, we are the first to isolate and characterize cell populations according to ALDH activity and gene expression of ALDH1A isoforms in adult articular tissues, though a number of studies on ALDH1A2 expression were performed in developing embryos of different vertebrate species. For characterization of ALDHþ and ALDH populations, in addition to traditional markers of chondrogenesis, we also included genes of Melanoma Inhibitory Activity protein (MIA), an important regulator of chondrogenic differentiation and cartilage maintenance15, required for properly ultrastructurally ordered collagen fiber composition16; antitrypsin A1 (SERPINA1) e the marker for chondrogenic differentiation17, and protein lubricin (PRG4), synthesized by articular cartilage surface chondrocytes. For further investigation of those populations, we employed a panel of genes of surface markers, characteristic of chondrocytes and mesenchymal stromal cells (MSCs). In addition, expression of some genes, characteristic of stem cells, NANOG, OCT4 and SOX2, as well as ABC transporter ABCG2, characteristic of side population phenotype, was also analyzed in those cells.

Methods Chondrocyte isolation and culture Articular cartilage was obtained from the knee joints during the articular replacement surgery at Republican Vilnius University Hospital, according to the Study Protocol, approved by the Lithuanian Bioethics Committee, with informed consents of the patients. Samples were collected from 26 patients, 52e79 years old, mean age 67.5 ± 7.6; males e 5, females e 21, with OA grade 2e4, mean 2.9 (according to DL Holden classification18). Age, gender and OA grade of patients is presented in Supplement 1. Chondrocytes were isolated as described elsewhere19. Briefly, cartilage specimens were cut from areas without visible lesions, weighed, diced to 5 mm2 pieces, incubated overnight in DMEM with 1% antibiotics (amphotericin, penicillin and streptomycin, Biological Industries, Israel) in incubator (humidified atmosphere containing 5% CO2, 37 C), then digested for 60 min with 26.5 U/ml pronase (Sigma), dissolved in DMEM, in the same conditions. After the digestion cartilage pieces were washed with PBS, finely minced and digested with 545 U/ml of collagenase type II (Biochrom, Germany) in DMEM for 3 h at 37 C with shaking. The resulting cell suspension was filtered through a 20 mm nylon net filter (Millipore, Ireland), washed twice with PBS, counted with hemocytometer and seeded at density (20,000e30,000 cells/cm2) in tissue culture flasks in growing medium (Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (Biochrom, Germany) and 1% antibiotics) and cultured in incubator. Subconfluent cells were harvested by trypsin/EDTA digestion and replated at a density of 6600 cells/cm2. The medium was changed twice a week. Evaluation of ALDH activity and cell sorting Isolated chondrocytes after cultivation in monolayer (culture duration from 1 day to 12 weeks) were trypsinized, washed with PBS and analyzed with Aldefluor™ assay (StemCell technologies, Canada) according the protocol provided by manufacturer. Incubation with Aldefluor™ reagent was performed for 60 min at 37 C, then washed and resuspended in fresh cold Aldefluor™ buffer, placed on ice and immediately analyzed on flow cytometer/ sorter FACSAria (Becton Dickinson, USA) following manufacturer's recommendations. An aliquot of cells was incubated under identical conditions with specific ALDH inhibitor e diethylaminobenzaldehyde (DEAB) as a negative control. Data were analyzed with BD FACSDiva software. Cells that were found in ALDHþ gate were named ALDHpos, and cells in ALDH gate were named ALDHneg cells. Representative plots of flow cytometric analyses and gating examples are presented in Fig. 1. To evaluate the differences between ALDH-expressing and not expressing chondrocytes, they were FACS-sorted according ALDH activity [Fig. 1(D)]. Cells, sorted to ALDHþ fraction were named ALDHþ cells and ones sorted to ALDH fraction e ALDH cells. In a few experiments intermediate ALDHdim fraction was also collected. Part of sorted cells were washed in PBS, immediately frozen in liquid nitrogen and stored at 80 C for gene expression analysis. Another part was seeded in culture flasks in growth medium for further culture or colony forming (CFU) analysis. Cell surface marker coexpression with ALDH activity Cells were processed for ALDH activity analysis, as described above, washed and resuspended in fresh cold Aldefluor buffer, stained with specific antibodies for 30 min on ice, in the dark, washed again, then resuspended in fresh cold Aldefluor buffer and


875

Fig. 1. Typical Aldefluor™ fluorescence plots for flow cytometric analysis of ALDH activity and cell sorting. A e Side scatter (SSC) vs forward scatter (FSC) plot, analyzed population marked as P1. B e Control cells, incubated with Aldefluor™ reagent þ DEAB. Gates ALDH and ALDHþ were created for ALDH activity in presence of DEAB (about 3% in ALDHþ gate). C: Cells, incubated with Aldefluor™ reagent; activity expressed in percent of cells in ALDHþ gate, (control, the 3%, was subtracted afterwards); D: plot and gates for sorting ALDH and ALDHþ cells. Intermediate fraction named ALDHdim was also collected in some experiments.

analyzed on FACSAria. Non-specific staining was assessed using fluorochrome-, isotype- and species-matched isotype controls. All monoclonal antibodies and isotype controls used in this study (Table I) were directly conjugated to fluorochromes. Gates for ALDH activity were placed for control sample with DEAB, as described above, but in histogram plot [Fig. 3(A)], and gates for surface markers were placed for the appropriate isotype controls. Coexpression data were analyzed as ratio of mean fluorescence intensity (MFI) value of labeled surface markers in gate ALDHþ vs the value in gate ALDH. Table I Antibodies used for surface marker analysis Antibody

Isotype Clone

Producer

CD29-APC (allophycocyanin) CD44 FITC (Fluorescein isothiocyanate) CD46 FITC CD47-PE (R-Phycoerythrin) CD49a-PE CD49c-PE CD49e-PE CD54-APC CD55-PE

IgG1 IgG2b

MEM-101A C-26

EXBIO BD Pharmingen

IgG2b IgG1 IgG1 IgG1 IgG1 IgG1 IgG2a

CD56-APC CD63-PE CD73-PE CD90 FITC CD105-APC CD140b-APC CD146-APC CD166-PE CD349- Alexa fluor 647 Notch1-APC MSCA-1- APC Isotype control-PE

IgG2b IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgM IgG1 IgG1 IgG1

3F1 B6H12 SR84 C3II IIA1 51-10C9 NaM-164D3 B159 H5C6 AD2 5E-10 SN6 18A2 P1H12 3A6 W3C4E11 MHN-519 W8B2 e

Isotype control-PE Isotype control-APC

IgG2a IgG1

MOPC-173 e

Isotype control-APC Isotype control-Alexa fluor 647

IgG2b IgM

e MM-30

BD Pharmingen BD Pharmingen BD Pharmingen BD Pharmingen BD Pharmingen BD Pharmingen Santa Cruz Biotech BD Pharmingen BD Pharmingen BioLegend BioLegend eBioscience BioLegend BD Pharmingen BioLegend BioLegend eBioscience Miltenyi Biotec Santa Cruz Biotech Biolegend Santa Cruz Biotech Invitrogen BioLegend

RNA extraction and RT-qPCR RNA extraction from harvested chondrocytes was performed with RNeasy Mini Spin columns (Qiagen) according to the manufacturer's instructions. RNA concentration and purity were

measured with the NanoPhotometer™ Pearl (Implen). RNA samples were treated with DNase I (Fermentas, Lithuania) and cDNA synthesis was performed with the Maxima®First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer's protocols. PCRs were performed using Maxima® Probe qPCR Master Mix (2) (Fermentas) and Stratagene MX-3005P detection instrument (Agilent Technologies). The TaqMan® Gene Expression Assays (Applied Biosystems) for 19 genes were used for gene expression analysis (Table II). The PCR reaction volume was 25 ml with 0.5 ml of 20 Taqman® Gene Expression Assay mix. All reactions were run in triplicates. Cycle conditions were as follows: initial denaturation step for 10 min at 95 C, followed by 40 cycles of 15 s at 95 C for denaturation and 60 s at 60 C for annealing and extension. Each RNA sample was controlled for genomic DNA contamination by reactions without reverse transcriptase (RT-) and reagent contamination was checked by the reactions without template (NTC). Relative mRNA levels were expressed as 2DCt and the fold change in gene expression in sorted fractions (ALDHþ vs ALDH) was expressed as 2DDCt.

Table II The TaqMan® Gene Expression Assays used for gene expression analysis in chondrocytes Gene, assay ID

Encoded protein

RPS9 Hs02339424_m1 COL2A1 Hs01060345_m1 COL10A1 Hs00166657_m1 SOX9 Hs00165814_m1 SOX2 Hs01053049_s1 SERPINA1 Hs00165475_m1 MIA Hs00197954_m1 PRG4 Hs00981633_m1 Runx2 Hs00231692_m1 PPARG Hs01115513_m1 NANOG Hs 02387400_g1 ALDH1A1 Hs00946916_m1 ALDH1A2 Hs00180254_m1 ALDH1A3 Hs00167476_m1 ALDH2 Hs01007998_m1 BGLAP Hs 01587814_g1

40S ribosomal protein S9 (housekeeping gene) Collagen type II, alpha 1 Collagen type X, alpha 1 Transcription factor SOX-9 Transcription factor SOX-2 Alpha-1 antiproteinase, antitrypsin

ALPL Hs01029144_m1 OCT4 Hs00999632_g1 ABCG2 Hs01053790_m1

Melanoma inhibitory activity Proteoglycan 4, lubricin Runt-related transcription factor 2 Peroxisome proliferator-activated receptor gamma Transcription factor NANOG Aldehyde dehydrogenase 1 family, member A1 Aldehyde dehydrogenase 1 family, member A2 Aldehyde dehydrogenase 1 family, member A3 Aldehyde dehydrogenase 2 (mitochondrial) Bone Gamma-Carboxyglutamate Protein, Osteocalcin Alkaline Phosphatase Octamer-binding transcription factor 4 ATP-binding cassette, sub-family G, member 2

876


C

100 80

AGE, years

60 pos

ALDH

40

,%

20 0

60 50

ALDHpos,%

A

40 30 20 10 0

F/2 F/2 F/2 F/2 F/3 F/3 F/4 M/3 M/4

1-6 d

1-2 w

3-4 w

5-8 w

12 w

76 M/3 77 F/2 72 F/3 65 F/2 52 F/2 65 F/3 65 F/2 57 F/4 72 F/3

gender/OA grade

B

60

ALDHpos,%

40 20 0

1-6 days

2 weeks

3-4 weeks

5-8 weeks

12 weeks

n=9

n=9

n=12

n=4

n=2

-20 -40 Fig. 2. A e Distribution of ALDHpos cells according patients' age, gender and OA grade (according to DL Holden OA grade classification). Lines with error bars represent mean and 95% CI. B e Amount of ALDH-expressing cells (ALDHpos) in freshly isolated and in monolayer cultured chondrocytes, expressed as percent of P1 population. Dots in the scatter plot represent mean value for every different patient, line with error bars represent mean of all patients and 95% CI. n is number of different patients analyzed. C e Percentage of ALDHpos cells during cultivation for individual cell lines, marked as patient age and gender/OA grade.

Statistical analysis Data were analyzed using GraphPad Prism 5 software and paired samples t-test was used on dCt values of ALDHþ and ALDH cells. A ratio t-test was used for ALDH activity and surface marker co-expression analysis on log-transformed MFI values. P values less than 0.05 were considered statistically significant. Results ALDH activity and surface marker co-expression When chondrocytes were analyzed 1e6 days after isolation, big portion of cells (up to 50%) showed high ALDH activity, which was blocked by specific inhibitor DEAB [Fig. 1(B) and (C)]. Percentage of ALDHpos cells, measured at the earliest time point, 1e6 days [Fig. 2(A)], did not show any obvious dependence on patients' age (in the analyzed range), gender and OA grade. During incubation in monolayer the percentage of ALDHpos cells gradually declined. Data for chondrocytes, evaluated from 1 day to 12 weeks of cultivation are presented in Fig. 2(B). Estimation of ALDH activity during culture for the cells, isolated from the same patient, showed similar trend [Fig. 2(C)]. To analyze specific immunophenotype, characteristic of the cells with ALDH activity, we performed combined staining for Aldefluor and cell surface markers for cells, cultivated in monolayer during 4e21 days (Fig. 3). Coexpression data analysis revealed that many of tested markers were slightly stronger expressed in ALDHpos than ALDHneg cells. Markers, attributed to this group included: CD29, CD49a, CD49c, CD105, CD349 and Notch1. The ratio of MFI for tested surface markers is presented in Fig. 3(B). Expression of CD54, and CD55 was significantly stronger expressed in ALDHpos cells [Fig. 3(C)]. Two markers were distributed equally between ALDHpos vs ALDHneg populations: CD56, and CD63 [Fig. 3(D)]. Some markers showed tendency to be stronger expressed in ALDHneg fraction [Fig. 3(E)]. This group included CD47, CD140b, CD146 and CD166.

ALDH-sorted cells: ALDH activity, surface marker expression and CFU forming Chondrocytes cultured for 2e3 weeks were labeled with Aldefluor™ and sorted with FACSAria into ALDHþ and ALDH fractions [Fig. 1(D)]. ALDH/dim/þ fractions of the sorted cells contained about 45%/30%/25% cells, respectively. After 1-day cultivation in culture flasks, aliquots of the seeded cells from all three fractions were collected, labeled again, and ALDH activity was measured. Results are presented in Fig. 4(A). About 50% of ALDHþ fraction was ALDH-positive cells. ALDH fraction had no ALDH activity, as expected. Intermediate ALDHdim fraction had some activity, but much less than ALDHþ fraction (20%). When ALDH was evaluated in the same sorted groups after 10-day cultivation in monolayer, the activity did not differ very much among them [Fig. 4(B)]. ALDHdim fraction did not change considerably, but activity in ALDHþ fraction dropped remarkably, while in ALDH it increased. The same measurements, performed for other sortings from different donors gave very similar results [Fig. 4(C)]: isolated ALDH-expressing cells did not keep this feature during 2e3-week cultivation in monolayer, and separated fractions became quite similar in terms of this characteristic. Combined staining for surface markers and ALDH activity in ALDH-sorted chondrocytes, performed 1 day after sorting, confirmed that expression of ALDH and CD55 may correlate [Fig. 5(A)]. Almost all cells in ALDHþ fraction expressed CD55. Some CD55-negative cells appeared in ALDHdim fraction, and much more of them were present in ALDH fraction; and again, after 2-week cultivation in monolayer, no differences for this marker expression were found among the fractions [Fig. 5(B)]. A panel of other surface markers was analyzed in isolated ALDHþ and ALDH cells from three donors. After cultivation for 2 weeks no noticeable and reproducible differences were identified between the two groups (not shown). CFU counts from both fractions were also similar (not shown).


877

Fig. 3. A e Gate, defining ALDHneg and ALDHpos populations for coexpression analysis (Control cells, incubated with Aldefluor™ reagent þ DEAB). B e Ratio of MFI value for every surface marker measured in gate ALDHþ vs value in gate ALDH. Error bars represent 95% CI; Statistical significance is depicted above the scatterplots (P values exceeding but close to 0.05 are shown in brackets). C-E e Flow cytometry plots for co-expression of ALDH activity with surface markers. Three groups were identified: C e surface markers, stronger expressed on ALDHpos cells, than ALDHneg cells; D e nearly equal distribution between ALDHpos and ALDHneg cells; and E e surface markers, stronger expressed on ALDHneg cells than ALDHpos cells.

ALDH-sorted cells: gene expression analysis Expression of genes was analyzed in ALDH-sorted fractions (Fig. 6). Results confirmed that cells in ALDHdim fraction had intermediate expression of all genes tested [Fig. 6(A) and (B)], as it was demonstrated for ALDH activity [Fig. 4(A)]. No different expression was found in ALDHdim fraction; therefore it can be considered as a mixture of ALDHþ and ALDH fractions and can

be omitted from further evaluation. Chondrocyte-specific genes, namely: main transcription factor SOX9, aggrecan (ACAN), collagen COL2A1, MIA, SERPINA1, and PRG4 were strongly expressed in 2-week cultured cells. Transcription factors of osteogenic and adipogenic phenotypes, RUNX2 and PPARG, respectively, and especially e marker of hypertrophic chondrocytes, COL10A1, were only slightly expressed. Markers of undifferentiated stem cells e NANOG, OCT4 and ABCG2 were very weakly, but stably expressed in

878


Fig. 4. Plots for ALDH activity in ALDH-sorted, then cultured 1 day after sorting (A) and 10 days after sorting (B) cells. Numbers inside the gates indicate percentage of ALDH-positive cells. C e percent of ALDH-expressing cells in ALDHþ and ALDH sorted fractions 2e3 weeks after sorting. Dots in the scatter plot represent different patients' cell lines (n ¼ 5), error bars represent 95% CI.

Fig. 5. Expression of surface markers CD55 on ALDH-sorted cells 1 day after sorting (A) and 10 days after sorting (B). Numbers inside the gates indicate percentage of CD55-positive cells.


all tested chondrocyte aliquots (2DCt values for ALDHþ and ALDH fractions in several experiments were similar to those showed for one representative measurement in Fig. 6(A) and (B), but are not shown). The gene expression ratio for ALDHþ vs ALDH sorted cells is presented in Fig. 6(C)e(E). Significantly higher expression of COL2A1 (up to 24-fold), as well as other chondrogenic genes e SOX9 and SERPINA1 were determined in ALDHþ fraction. The transcripts of COL1A1 and ACAN genes were very abundant, but the level did not differ between the fractions. Markers of multipotency e NANOG, ABCG2, OCT4 and SOX2 did not differ significantly between the populations. The only two genes, expressed stronger in ALDH fraction (up to 10-fold), were osteogenic markers RUNX2 and osteocalcin (BGLAP), though not statistically significant. Interestingly, another marker for osteogenic lineage (as well as stem cell marker in undifferentiated embryonic stem cells) e alkaline phosphatase (ALPL), did not differ between sorted cells. Our experiments revealed that isozyme ALDH1A1 was extremely weakly expressed (on the limit of detection level) in freshly isolated and cultured chondrocytes. Other isoforms of retinoic acid producing enzymes ALDH1A2 and ALDH1A3 were quite strongly expressed and differed significantly between sorted fractions. On the contrary, mitochondrial isoform ALDH2, that is also capable to activate Aldefluor™ 14, was also remarkably expressed, but only slightly differed between the populations. These data show that retinaldehyde dehydrogenases ALDH1A2 and ALDH1A3, at least in part, are responsible for the fractions' separation. Discussion In a number of studies on ALDH expression in mammalian tissues quantity of active cells fell between 0.2% for murine brain tissue, to 4% for human muscle mononucleated cells5,8,9, while 41e42% of ALDHpos cells have been detected in human adiposederived MSCs and dermal fibroblasts20. Further studies revealed direct involvement of ALDH1A isoenzymes in adipose tissue development and function11. Similarly, we found from 30 to 50%, of ALDH-positive cells in all samples of freshly isolated chondrocytes from macroscopically undamaged part of OA cartilage. During cultivation in monolayer, ALDH activity gradually declined, correlating in time with well known phenomenon of chondrocyte dedifferentiation in monolayer conditions. When cells were sorted according to ALDH activity and then cultured in monolayer separately, the differences in this activity, as well as in surface marker expression, disappeared in several days. This suggests that different ALDH expression does not represent specific population of cells in cartilage, but rather some functional properties, which change during culture. CFU counts in the both ALDH-sorted fractions were similar, thus ALDH activity seems unassociated with chondrocyte colony forming capacity either. For evaluation of differences between ALDHþ and ALDH cells in cartilage, we focused on gene expression analysis in the cells frozen immediately after sorting. This analysis revealed that ALDHþ cells expressed chondrogenic markers significantly stronger than those in ALDH fraction. Whereas, neither expression of dedifferentiated fibrotic or hypertrophic chondrocyte markers, COL1A1 and COL10A1 respectively, nor that of stem cell markers, namely OCT4, NANOG, SOX2 and ABCG2 differ between the two fractions. Therefore, ALDH activity looks like another marker, characteristic of collagen type II-producing chondrocytes. Immunophenotype analysis of ALDH-expressing cells revealed that CD54 and CD55 were the most strongly expressed surface markers on ALDH-containing cells. CD54 or Intercellular Adhesion Molecule-1 has been characterized as one of MSC markers21,

879

though its function in these cells is not known. In experiments with isolated and cultured human articular chondrocytes, downregulation of CD54 expression according to the dedifferentiation status was shown and the most appropriate combination of the ratio of Col II/Col I was found for CD54/CD44 expression22. This MFI ratio of CD54/CD44 was proposed to be the index of the chondro-differentiation status. Coexpression of ALDH activity with CD54bright in our experiments suggests that ALDH can be also associated with chondrogenic phenotype. CD55 or Complement decay-accelerating factor regulates the complement system and prevents the formation of a membrane attack complex on the cell surface, thus protecting cells from complement-mediated lysis. Complement expression was detected in human articular cartilage and presence of complement-activated membrane attack complex was shown in OA cartilage specimens23, illustrating the importance of cell protection from possible damage. Superior ALDH activity in the cells with the highest CD55 expression indicates that this activity is expressed in the cells that are better protected and are more likely to survive in inflammatory environment. The analysis of periodontal ligament derived cells showed that increased expression of transcripts for RUNX2 and CD166 was characteristic of highly osteogenic clones during their differentiation towards osteoblast phenotype24. We found that ALDHpos cells had almost no CD166. Gene expression analysis also revealed that osteogenic markers were stronger expressed in ALDH, than ALDHþ population. Hence, population with strong ALDH activity seems to be depleted from cells committed to osteogenic differentiation. We showed that chondrocytes had almost no isotype ALDH1A1, while the expression of ALDH1A2 and ALDH1A3 genes was strong, thus it is very likely that cells were sorted to ALDHþ and ALDH fractions according to activity of latter two isozymes. The differences of ALDH2 expression in those two fractions were very small and insignificant; therefore it could hardly participate in fraction separation and be responsible for different characteristics of fractioned cells. As this enzyme is not involved in ATRA synthesis, but rather in alcohol metabolism, we can assume that differences in the two fractions are associated with the differences in retinoid signaling. ATRA e the most important product of ALDH1 family enzymes plays a critical role in embryogenesis and function of vital organs in adult organisms. Studies with murine knockouts of ALDH1A enzymes showed that only embryos with ALDH1A2 knockouts died early in development due to severe defects in different processes, including development of the trunk and limbs25. When gene expression of primitive cartilage-committed progenitors of developing human embryo were compared to resting periarticular chondrocytes (immature definitive chondrocytes), among several thousand of genes, strongly enriched in human cartilage progenitors, several were associated with ATRA synthesis and transportation to nucleus e ALDH1A2 (enriched 29 times), retinol dehydrogenase 10, RDH10 (enriched 14 times), and cellular retinoic acid binding protein 1, CRABP1, which was expressed 58 times stronger (Table S2)26. Therefore, stronger ALDH activity could in some way be associated with the cartilage progenitors in developing embryo. In our experiments, up to 50% of freshly isolated OA cartilage cells had strong ALDH activity, therefore, it is not likely that all of them could be chondroprogenitors. Experiments with murine embryonic limb cell and organ cultures showed that ALDH1A2 was abundantly expressed both on gene and protein levels in early limb development stages and preceded cartilaginous formations, but then was totally excluded from developing cartilage as soon as cells started to express COL2A1. ALDH1A2 expression was inversely proportional to SOX9 as

880


Fig. 6. Gene expression in ALDH-sorted chondrocytes. A and B e one representative plot for sorted cells, when ALDHdim fraction was also collected. Results are expressed as 2DCt for every fraction separately, normalized to reference gene RPS9 expression; A e genes, related to chondrocyte, osteo- and adipogenic phenotype and stem cells. B eALDH1A and ALDH2 gene expression, ND e not detected. C-E e gene expression ratio for ALDHþ vs ALDH sorted chondrocytes (2DDCt); C e genes, related to chondrocyte phenotype. D e genes, related to osteo-/adipogenic phenotype and stem cells. E e gene expression of four ALDH isoforms. Box plots with whiskers represent interquartile range with median. Statistical significance is depicted above the box plots. n e Number of patients.

determined by qPCR27. In contrast, our results showed that chondrocytes with the highest ALDH activity were strongly and significantly enriched in COL2A1 expression, as well as other chondrogenic markers e SOX9 and SERPINA1. It was demonstrated that expression of ALDH1As and other retinoids in brain tissue differed remarkably between fetal and adult tissue28. It is very likely that ALDH expression and ATRA signaling differs in embryonic and adult cartilage as well. Further studies, especially with healthy cartilage, are needed to explain this phenomenon. In the majority of adult tissues analyzed so far all three isoforms of ALDH1A are expressed. In adult organisms ALDH1A1 is dominating isoform in kidney, liver, leukocyte, adipose tissue and contributes substantially to ATRA biosynthesis in many other tissues11,28. ALDH1A1 expression levels in adipocytes have been directly associated with obesity, glucose intolerance and inflammation. Recent findings demonstrate ALDH1A1, predominant isoform in bone, involvement in bone mass gain/loss and marrow adiposity29. Abnormally high ALDH1A3 expression in pancreas has been linked to decreased insulin secretion in diabetic mouse models. ALDH1A3 controls differentiation in mammary epithelia and dysregulation of this enzyme was associated with breast cancer11,13. Overexpression of ALDH1A1 and ALDH1A3 was also found in several other cancers with high ALDH activity30. In contrast to

other tissues, we found extremely low expression of ALDH1A1 in chondrocytes. While studies with ALDH1A2 expression during embryogenesis are abundant, relatively little has been done in adult organisms. In the analyzed adult human tissues ALDH1A2 had widespread expression, but lower than other isoforms. The highest expression of this isozyme was found in testis and ovary28. It was demonstrated that spinal cord injury in rats induced ALDH1A2 expression in polydendrocytes e activated oligodendrocyte and astrocyte precursors, while in uninjured spinal cord only low levels of the enzyme was detected in these cells31. Association of ALDH1A2 with regeneration was also found in other tissues and organisms. Comparative analysis of Zebrafish regeneration response to amputation/injury of adult caudal fin, adult heart, and larval fin revealed that ALDH1A2 was the first and one of the most highly induced genes. ALDH1A2 expression was critical for the formation of wound epithelium and blastema32. In urodele amphibians (that are capable of organ regeneration) additional treatment with retinoids after limb amputation caused “superregeneration” e a concentration-dependent increase in the amount of regenerated tissue, and often two regenerates appeared instead of one (reviewed in33). In experiments with murine partial hepatectomy ATRA signaling was involved in normal liver regeneration as the gene expression of ALDH1A2, CRABP1, and cellular retinol


binding protein 1 (CRB1) was induced 1.5 days after hepatectomy during the hepatocyte proliferation34. There is a lot of controversial data about the effect of retinoids in articular cartilage; a number of studies showed tissue degradation or reduced chondrogenic differentiation by ATRA treatment, while others revealed beneficial effects, especially in inflammatory arthritis35,36. The differential gene expression analysis in macroscopically intact part of OA articular cartilage vs osteophytic one from the same respective joint identified that ALDH1A2 was 24 times more enriched in the unaffected articular cartilage vs osteophytic cartilage, and this was the strongest enrichment of all genes analyzed37. This data suggest strong retinoid signaling in cartilage. Our results imply association of ALDH activity with phenotype of cartilage extracellular matrix producing cells. Whether ALDH activity and the demonstrated isotype composition is a characteristic of every cartilage, only articular cartilage, or injured/regenerating cartilage, still needs to be elucidated. Additional investigation is required for better understanding of the role that ALDH1A isoforms and other retinoid signaling participants play in cartilage homeostasis, degradation or regeneration. Conclusions We showed for the first time that a high percentage of human OA chondrocytes have high ALDH activity with stronger expression of surface markers CD54 and CD55, while that frequency is unlikely to correspond to the progenitor population in cartilage. Isotypes ALDH1A2 and ALDH1A3, involved in retinoic acid signaling, are responsible, at least in part, for ALDH activity, measured by Aldefluor™. Chondrogenic markers including COL2A1, SOX9 and SERPINA1 are significantly enriched in ALDH-active cells from the cartilage. Therefore, ALDH activity and expression of ALDH1A2 or ALDH1A3 isotypes are likely to serve as markers of active chondrocytes with enriched production of collagen type II in human adult articular cartilage, at least during OA. Authors' contributions AU: design of the study, collection and analysis of flow cytometry data, cell sorting, manuscript writing and editing. EBe: contribution to design of the study, critical revision, contribution to manuscript writing and editing. EBa: contribution to design of the study, collection and analysis of gene expression data, statistical expertise, contribution to manuscript writing and editing. SG: chondrocyte isolation, cell cultures, contribution to flow cytometry analysis, sorting and manuscript writing. NP: enrollment and evaluation of patients and cartilage sample collection, supervision of bioethics requirements, and contribution to manuscript editing. CJ: Obtaining of funding, contribution to design of the study, critical revision and final approval of the manuscript. Competing interest statement The authors declare no conflicts of interest. Role of the funding source Study was funded by the European Community's Seventh Framework Program (FP7/2007-2013) the collaborative project grant No: HEALTH-F5-2010-241719 ADIPOA “Adipose-derived stromal cells for osteoarthritis treatment”. Acknowledgments The authors would like to thank Virginija Plungyte, Ilona Mak te, Saule Valiu niene and simova, Giedre Miskinyte, Rugile Juskevi ciu

881

Jaroslav Denkovskij for technical assistance. We thank the surgeons and nursing staff in Republican Vilnius University Hospital for facilitating the harvest of patient tissue for this research. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.joca.2015.11.019. References 1. Storms RW, Trujillo AP, Springer JB, Shah L, Colvin OM, Ludeman SM, et al. Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity. Proc Natl Acad Sci USA 1999;96:9118e23. 2. Armstrong L, Stojkovic M, Dimmick I, Ahmad S, Stojkovic P, Hole N, et al. Phenotypic characterization of murine primitive hematopoietic progenitor cells isolated on basis of aldehyde dehydrogenase activity. Stem Cells 2004;22:1142e51. 3. Hess DA, Meyerrose TE, Wirthlin L, Craft TP, Herrbrich PE, Creer MH, et al. Functional characterization of highly purified human hematopoietic repopulating cells isolated according to aldehyde dehydrogenase activity. Blood 2004;104: 1648e55. 4. Putman DM, Liu KY, Broughton HC, Bell GI, Hess DA. Umbilical cord blood-derived aldehyde dehydrogenase-expressing progenitor cells promote recovery from acute ischemic injury. Stem Cells 2012;30:2248e60. 5. Vauchez K, Marolleau JP, Schmid M, Khattar P, Chapel A, Catelain C, et al. Aldehyde dehydrogenase activity identifies a population of human skeletal muscle cells with high myogenic capacities. Mol Ther 2009;17:1948e58. 6. Jean E, Laoudj-Chenivesse D, Notarnicola C, Rouger K, Serratrice N, Bonnieu A, et al. Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells. J Cell Mol Med 2011;15:119e33. 7. Vella JB, Thompson SD, Bucsek MJ, Song M, Huard J. Murine and human myogenic cells identified by elevated aldehyde dehydrogenase activity: implications for muscle regeneration and repair. PLoS One 2011;6:e29226. 8. Roehrich ME, Spicher A, Milano G, Vassalli G. Characterization of cardiac-resident progenitor cells expressing high aldehyde dehydrogenase activity. Biomed Res Int 2013;2013: 503047. 9. Corti S, Locatelli F, Papadimitriou D, Donadoni C, Salani S, Del Bo R, et al. Identification of a primitive brain-derived neural stem cell population based on aldehyde dehydrogenase activity. Stem Cells 2006;24:975e85. 10. Vasiliou V, Thompson DC, Smith C, Fujita M, Chen Y. Aldehyde dehydrogenases: from eye crystallins to metabolic disease and cancer stem cells. Chem Biol Interact 2013;202:2e10. 11. Petrosino JM, Disilvestro D, Ziouzenkova O. Aldehyde dehydrogenase 1A1: friend or foe to female metabolism? Nutrients 2014;6:950e73. 12. Gudas LJ. Emerging roles for retinoids in regeneration and differentiation in normal and disease states. Biochim Biophys Acta 1821;2012:213e21. 13. Napoli JL. Physiological insights into all-trans-retinoic acid biosynthesis. Biochim Biophys Acta 1821;2012:152e67. 14. Moreb JS, Ucar D, Han S, Amory JK, Goldstein AS, Ostmark B, et al. The enzymatic activity of human aldehyde dehydrogenases 1A2 and 2 (ALDH1A2 and ALDH2) is detected by Aldefluor, inhibited by diethylaminobenzaldehyde and has significant effects on cell proliferation and drug resistance. Chem Biol Interact 2012;195:52e60.

882


15. Tscheudschilsuren G, Bosserhoff AK, Schlegel J, Vollmer D, Anton A, Alt V, et al. Regulation of mesenchymal stem cell and chondrocyte differentiation by MIA. Exp Cell Res 2006;312: 63e72. 16. Moser M, Bosserhoff AK, Hunziker EB, Sandell L, Fassler R, Buettner R. Ultrastructural cartilage abnormalities in MIA/CDRAP-deficient mice. Mol Cell Biol 2002;22:1438e45. 17. Boeuf S, Steck E, Pelttari K, Hennig T, Buneb A, Benz K, et al. Subtractive gene expression profiling of articular cartilage and mesenchymal stem cells: serpins as cartilage-relevant differentiation markers. Osteoarthritis Cartilage 2008;16:48e60. 18. Holden DL, James SL, Larson RL, Slocum DB. Proximal tibial osteotomy in patients who are fifty years old or less. A longterm follow-up study. J Bone Joint Surg Am 1988;70:977e82. 19. Maumus M, Manferdini C, Toupet K, Peyrafitte JA, Ferreira R, Facchini A, et al. Adipose mesenchymal stem cells protect chondrocytes from degeneration associated with osteoarthritis. Stem Cell Res 2013;11:834e44. 20. Blasi A, Martino C, Balducci L, Saldarelli M, Soleti A, Navone SE, et al. Dermal fibroblasts display similar phenotypic and differentiation capacity to fat-derived mesenchymal stem cells, but differ in anti-inflammatory and angiogenic potential. Vasc Cell 2011;3:5. 21. Calloni R, Cordero EA, Henriques JA, Bonatto D. Reviewing and updating the major molecular markers for stem cells. Stem Cells Dev 2013;22:1455e76. 22. Hamada T, Sakai T, Hiraiwa H, Nakashima M, Ono Y, Mitsuyama H, et al. Surface markers and gene expression to characterize the differentiation of monolayer expanded human articular chondrocytes. Nagoya J Med Sci 2013;75: 101e11. 23. Wang Q, Rozelle AL, Lepus CM, Scanzello CR, Song JJ, Larsen DM, et al. Identification of a central role for complement in osteoarthritis. Nat Med 2011;17:1674e9. 24. Saito MT, Salmon CR, Amorim BR, Ambrosano GM, Casati MZ, Sallum EA, et al. Characterization of highly osteoblast/cementoblast cell clones from a CD105-enriched periodontal ligament progenitor cell population. J Periodontol 2014;85: e205e211. 25. Niederreither K, Subbarayan V, Dolle P, Chambon P. Embryonic retinoic acid synthesis is essential for early mouse postimplantation development. Nat Genet 1999;21:444e8. 26. Wu L, Bluguermann C, Kyupelyan L, Latour B, Gonzalez S, Shah S, et al. Human developmental chondrogenesis as a basis

27.

28.

29.

30.

31.

32.

33. 34.

35.

36.

37.

for engineering chondrocytes from pluripotent stem cells. Stem Cell Reports 2013;1:575e89. Hoffman LM, Garcha K, Karamboulas K, Cowan MF, Drysdale LM, Horton WA, et al. BMP action in skeletogenesis involves attenuation of retinoid signaling. J Cell Biol 2006;174: 101e13. Xi J, Yang Z. Expression of RALDHs (ALDH1As) and CYP26s in human tissues and during the neural differentiation of P19 embryonal carcinoma stem cell. Gene Expr Patterns 2008;8: 438e42. Nallamshetty S, Le PT, Wang H, Issacsohn MJ, Reeder DJ, Rhee EJ, et al. Retinaldehyde dehydrogenase 1 deficiency inhibits PPARgamma-mediated bone loss and marrow adiposity. Bone 2014;67:281e91. Pors K, Moreb JS. Aldehyde dehydrogenases in cancer: an opportunity for biomarker and drug development? Drug Discov Today 2014;19:1953e63. Kern J, Schrage K, Koopmans GC, Joosten EA, McCaffery P, Mey J. Characterization of retinaldehyde dehydrogenase-2 induction in NG2-positive glia after spinal cord contusion injury. Int J Dev Neurosci 2007;25:7e16. Mathew LK, Sengupta S, Franzosa JA, Perry J, La Du J, Andreasen EA, et al. Comparative expression profiling reveals an essential role for raldh2 in epimorphic regeneration. J Biol Chem 2009;284:33642e53. Maden M, Hind M. Retinoic acid, a regeneration-inducing molecule. Dev Dyn 2003;226:237e44. Liu HX, Ly I, Hu Y, Wan YJ. Retinoic acid regulates cell cycle genes and accelerates normal mouse liver regeneration. Biochem Pharmacol 2014;91:256e65. Ho LJ, Lin LC, Hung LF, Wang SJ, Lee CH, Chang DM, et al. Retinoic acid blocks pro-inflammatory cytokine-induced matrix metalloproteinase production by down-regulating JNK-AP-1 signaling in human chondrocytes. Biochem Pharmacol 2005;70:200e8. Kwok SK, Park MK, Cho ML, Oh HJ, Park EM, Lee DG, et al. Retinoic acid attenuates rheumatoid inflammation in mice. J Immunol 2012;189:1062e71. Gelse K, Ekici AB, Cipa F, Swoboda B, Carl HD, Olk A, et al. Molecular differentiation between osteophytic and articular cartilageeclues for a transient and permanent chondrocyte phenotype. Osteoarthritis Cartilage 2012;20:162e71.