Stem Cell Research 47 (2020) 101899 Contents lists available at ScienceDirect Stem Cell Research journal homepage: www.elsevier.com/locate/scr Extrinsic modulation of integrin α6 and progenitor cell behavior in T mesenchymal stem cells Nuria Nieto-Nicolaua,b,d, Raquel M. de la Torreb, Oscar Fariñasa,d, Andrés Savioa,d, ⁎ Anna Vilarrodonaa,d, Ricardo P. Casaroli-Maranoa,b,c,d, a Barcelona Tissue Bank (BTB) & Donor Center, Banc de Sang i Teixits (BST), Barcelona, Spain b CellTec-UB, University of Barcelona, Barcelona, Spain c Department of Surgery, School of Medicine & Hospital Clinic de Barcelona, University of Barcelona, Barcelona, Spain d Institute of Biomedical Research Sant Pau (IIB-Sant Pau), Barcelona, Spain A R T I C LE I N FO A B S T R A C T Keywords: Mesenchymal stem cells (MSC) are heterogeneous cells of complex nature that show different potentials while Mesenchymal stem cells different culture conditions can modify their functionalities through interactions with the microenviroment. Integrin alpha 6 Here, we found that bone marrow (BM) MSC from different donor sources and passages that expressed higher Clonogenicity levels of α6 integrin subunit (ITGA6), showed higher clonogenicity, migration and differentiation potential. Proliferation ITGA6 showed important roles improving these potentials and regulating proliferation through protein kinase B Protein kinase B (AKT) pathway and cell cycle inhibitor proteins p53 and p21. Moreover, ITGA6 downregulation impaired mi- Migration gration. Cell confluence regulated ITGA6, increasing its expression in low density cultures and decreasing in high density cultures. Besides, ITGA6- cells expressed ITGA6 when seeded at low densities. We found higher ITGA6 expression on fibronectin substrates at lower confluency. Fibronectin increased proliferation, clonogenicity, activation of AKT, decreased cell cycle inhibitor proteins and augmented growth factors expression. Spheres- derived MSC showed higher ITGA6 expression and enhanced potentials for migration, clonogenicity and pro- liferation. In conclusion, though there is an intrinsic regulation of ITGA6 expression, associated to the progenitor potential of BM-MSC, this expression is regulated by culture conditions and is translated in changes in cell behavior and proliferation. This knowledge could be used to enhance the potential of BM-MSC for clinical application. 1. Introduction phenotypic identification of MSC is based on the expression of CD73, CD90 and CD105 among other markers (Dominici et al., 2006) but none Mesenchymal stem cells (MSC) have emerged as a promising tool for of these markers could identify progenitor phenotypes. However, MSC tissue engineering because their capability to differentiate toward are a heterogeneous population of cells with different capabilities various lineages (Liu et al., 2009). These cells are also accessible so- (Colter et al., 2001) and MSC from different donors or somatic sources matic stem cells that can be easily isolated. Consequently, several can have different capacities (Alt et al., 2012; Zaim et al., 2012; Zhu clinical trials use MSC in disorders that need organ or tissue re- et al., 2009). These issues can affect therapeutic results. So, it is ne- generation. This is the case of osteogenesis imperfecta, bone fractures, cessary the search for markers that could distinguish the early pro- heart infarction, autoimmune disorders or graft-versus-host disease genitors of these cells and increase the knowledge of the intrinsic and (Squillaro et al., 2016). extrinsic mechanisms modulating MSC progenitor behavior. Therefore, MSC are defined by plastic adherence, multilineage differentiation the characterization of optimal culture conditions to enhance the po- potential and a mesenchymal phenotype (Dominici et al., 2006). The tential of MSC is of great significance. Abbreviations: a-MSC, bone marrow mesenchymal stem cells from alive healthy donors; AT-MSC, adipose tissue mesenchymal stem cells; BM-MSC, bone marrow mesenchymal stem cells; CFE, colony forming efficiency; D+1, day one of culture; D+7, day seven of culture; ed-MSC, mesenchymal stem cells from cadaveric encephalic death donors; FN, fibronectin; ha-MSC, bone marrow mesenchymal stem cells from cadaveric heart arrest donors; ITGA6, integrin alpha 6; LN, laminin- 111; P, passage number ⁎ Corresponding author at: Department of Surgery, School of Medicine, University of Barcelona, Calle Sabino de Arana 1 (2nd floor – ophthalmology), E-08028 Barcelona, Spain. E-mail address: rcasaroli@ub.edu (R.P. Casaroli-Marano). https://doi.org/10.1016/j.scr.2020.101899 Received 11 March 2020; Received in revised form 11 June 2020; Accepted 24 June 2020 Available online 30 June 2020 1873-5061/ © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). N. Nieto-Nicolau, et al. Stem Cell Research 47 (2020) 101899 Stem cells are directed by the extracellular microenvironment, 2.3. Spheroid and spheroid-derived MSC generation which modulates their self-renewal and differentiation. This requires a close interaction between stem cells and the signals arising from the For sphere formation, BM-MSC from healthy donors at passage 3–4 niche (Bara et al., 2014). In this sense, integrins binding to extracellular were plated at low density in ultralow-adherence 35 mm dishes matrix proteins can modulate stem cell behavior (Frith et al., 2012). (Invitrogen) in spheroid formation medium composed of DMEM/F12 Integrin alpha 6 (ITGA6) expression is conserved among different po- (1:1)/ human endothelial serum-free medium (1:2) (Invitrogen), 15% pulations of stem cells, such as embryonic stem cells, epithelial intestine chicken embryo extract (Seralab, Huissen, Netherland), 0.1 mM β- stem cells, neural stem cells and some MSC (Krebsbach and Villa-Diaz, mercaptoethanol (Sigma), 1% nonessential amino acids (Sigma), 1% N2 2017), and plays important functions regulating self-renewal (Villa- and 2% B27 supplements (Invitrogen), 20 ng/ml recombinant human Diaz et al., 2016) and stemness (Lee et al., 2009; Yu et al., 2012). The fibroblast growth factor (FGF)-basic, 20 ng/ml recombinant human interaction between microenvironmental signals and intrinsic cell cues epidermal growth factor (EGF), 20 ng/ml recombinant human platelet- are necessary for the preservation of progenitor potential and any derived growth factor (PDGF-AB), 20 ng/ml recombinant human on- shortcoming in this interaction is linked to a decrease in this potential. costatin M (227 aa OSM, 20 ng/ml) and 40 ng/ml recombinant human Increasing the knowledge between extrinsic and intrinsic cues would IGF-1 (all supplements from Peprotech) and maintained as described allow to improvements in the regenerative potential of MSC for cell (Isern et al., 2013). To generate spheroid-derived MSC, the MSC therapy. spheroids were seeded with MSC medium in adherent conditions, In this research, we aimed to determine whether the expression of grown to the end of the passage, trypsinized, and used for experiments. ITGA6 could be modified extrinsically and whether this fact can influ- ence the progenitor behavior of BM-MSC. To do so, we first considered 2.4. Multilineage differentiation experiments the intrinsic expression of ITGA6 in MSC from different donor sources among passages and evaluated the implications of this integrin on Differentiation experiments were performed as described before proliferation, differentiation, migration and clonogenicity. Then, we (Mosna et al., 2010). Adipogenesis was performed with adipogenic studied the extrinsic modulation of ITGA6 expression through different induction medium containing Dulbecco's Modified Eagle's Medium 1 g/ substrates in low- and high-density cultures, exploring the impact in cell l (DMEM, Invitrogen) supplemented with 0.5 mM isobutyl methyl- behavior. Finally, we explored a strategy to increase the expression of xanthine (Sigma-Aldrich, Munich, Germany), 1 µM dexamethasone ITGA6 and the potentials of MSC for cell therapy using MSC spheroids. (Sigma-Aldrich), and 200 µM indomethacin (Sigma-Aldrich) and con- Our results suggest the existence of not only an intrinsic regulation of firmed at 28 days by Oil-Red-O staining, eluted with 100% isopropanol. ITGA6 associated to the progenitor potential, but also a micro- Osteogenic induction was performed in DMEM 1 g/l medium with 1 µM environmental regulation of ITGA6 expression, that is translated in a dexamethasone, 50 µg/mL ascorbic acid (Sigma-Aldrich), and 10 mM modulation of cell cycle proteins, proliferation, differentiation, migra- glycerol 2-phosphate (Sigma-Aldrich) and confirmed at 28 days by tion and clonogenicity. Alizarin Red staining, eluted with 10% cetylpyridinium chloride. Cells (2 × 105 cells/cm2) were pelleted and chondrogenic induction was performed adding DMEM 1 g/l supplemented with 0.4% insulin-trans- 2. Materials and methods ferrin-selenium (ITS, Sigma-Aldrich), 350 µM L-proline (Sigma-Al- drich), 170 nM ascorbic acid, 1 mM sodium pyruvate (Lonza, Verviers, 2.1. Ethical considerations Belgium) and 10 ng/ml transforming growth factor-beta 1 (TGF-β1, Sigma-Aldrich) for 28 days. After that, pellets were fixed with 4% This study followed the ethical precepts of the Declaration of paraformaldehyde for 1 h at room temperature, stained with Alcian Helsinki (Fortaleza, Brazil, Oct 2013) and was approved by local ethics Blue, embedded in paraffin, and sectioned at thicknesses of 5 µm. committee (CEIC, Hospital Clinic de Barcelona; Ref 7365-12). Human samples were processed according to guidance for clinical use (EEC 2.5. Flow cytometry characterization and sorting regulations 2004/23/CE and 2006/17/CE) and to the legal require- ments Spain (Law 14/2007, RD 1716/2011 and RD 9/2014). Samples Cells were blocked with 10% FCS for 10 min and incubated for were obtained under informed consent. The use of personal data com- 30 min at room temperature with conjugated antibodies (Table S1). plied with local regulations (Law/05/2018). Samples were analyzed by FACS (Calibur Flow Cytometer, BD Bios- ciences, San José, CA, USA) and data were evaluated by Summit version 3.1 software (Cytomation, Fort Collins, CO, USA). ITGA6 analysis was 2.2. Isolation and culture of cells performed at the end of the passages, unless indicated and size analysis was performed as described before (Romano et al., 2003). Sorting was BM-MSC were obtained by iliac crest bone marrow aspiration, and performed in a FACS Aria Fusion cell sorting cytometer (BD Bios- transmissible infections were excluded by serologic analyses. BM-MSC ciences), and post-sorting vitality testing was done with trypan blue to were isolated, as described elsewhere (Rider et al., 2008). BM-MSC ensure equal survival of the sorted populations. Mouse monoclonal IgG were cultured in MSC medium comprising α minimum essential isotype antibody was used as a negative control (Table S1). To assay the medium with GlutaMAX™ (αMEM-GT, Invitrogen, Carlsbad, CA) sup- influence of different substrates on ITGA6 expression, cells were seeded plemented with non-inactivated 10% fetal calf serum (FCS, Invitrogen) at a density of 1,000 cells/cm2 on either fibronectin at 10 µg/mL (FN, and 1% antibiotics and maintained in 5% CO2 at 37 °C. Same numbers Sigma-Aldrich), laminin-111 at 20 µg/mL (LN, Sigma-Aldrich, L2020, of cells per donor were pooled at passage 1. Cells from 5 live healthy www.sigmaaldrich.com/catalog/product/sigma/l2020?lang=es& donors at passage 3 or 4 were used for experiments. When indicated, 3 region=ES) or plastic. Cells were grown for 24 h (D + 1, low density) different donor sources were used: alive healthy (a-MSC), cadaveric or 7 days (D + 7, high density). Cells were then trypsinized and subject heart arrest (ha-MSC), and cadaveric encephalic death donors (ed- to flow cytometry and clonogenic assay. MSC). Isolation, culture, and characterization of adipose tissue MSC (AT-MSC) was showed previously (Martínez-Conesa et al., 2012). 2.6. Colony forming efficiency assay Doubling population time was calculated as described before (Piera- Velazquez et al., 2002). Induced pluripotent stem cells (IPSC) were For clonogenic experiments, 10 cells/cm2 were seeded in 35 mm kindly gifted by Angel Raya and used for qPCR experiments. diameter plates and cultured in MSC medium for 14 days. After that, cells were stained with 0.5% crystal violet. Colonies were scored 2 N. Nieto-Nicolau, et al. Stem Cell Research 47 (2020) 101899 according to (Sotiropoulou et al., 2006). To test the influence of dif- above. Hoechst 33,342 (Sigma) was used for nucleus staining. Samples ferent substrates on the colony forming efficiency (CFE) potential, cells were mounted with Fluoromount (Sigma) and images were captured were seeded as described above. using a TCS SP2 Leica confocal laser scanning microscope (Leica Lasertechnick GmbH, Mannheim, Germany). Antibodies used are de- 2.7. Adhesion assay tailed in Table S1. For adhesion assays, 96 well plates were coated with specific ex- 2.12. Western blot analysis tracellular adhesion proteins, FN at 10 µg/ml, LN at 20 µg/ml or bovine serum albumin (BSA, Sigma) at 10 µg/ml as a control. We seeded 104 Western blot was performed as described before (Piera-Velazquez cells/cm2 on coated plates in MSC medium. After the indicated time, et al., 2002). Protein bands were revealed using an enhanced chemi- wells were stained with 0.5% crystal violet, washed, air dried, and luminescence substrate (Biological Industries, Reactiva, Barcelona, eluted with 33% acetic acid solution. Absorbance was measured with Spain) and recorded on autoradiography film (Kodak Rochester, NY, Tecan Infinite m200 pro (Tecan, Mannedorf). To test whether ITGA6 USA). Western blots were analyzed by digitally scanning the blots, and blocking could affect adhesion to the substrates, 5 × 103 cells were followed by densitometric analysis using ImageJ as indicated (Rasband, seeded with 10 µg/mL of an ITGA6 neutralizing antibody (Table S1) on 2012). All analyses were normalized to a tubulin loading control, ex- the coated or uncoated 96-well plates, and adhesion assay was per- cept for phosphorylated protein kinase B (p-AKT), which was normal- formed at 60 min. A mouse monoclonal IgG (Table S1) was used as a ized to the total AKT. control. 2.13. Extraction of mRNA and quantitative polymerase chain reaction 2.8. Migration assay Total RNA was extracted from cells using a PureLink RNA Mini Kit Cell migration was analyzed using 24-well inserts with 8 µm pore (Ambion, Invitrogen), following manufacturer’s instructions. The RNA size fluorescence blocking membranes (Corning, MA, USA) on 24-well concentration was measured using the Tecan Infinite m200 Pro, and plates. Cells were seeded in serum free medium (DMEM 1 g/l medium) 1 μg RNA was reverse-transcribed using Superscript III (Invitrogen), in the insert. Culture medium with 10% FCS was added to the lower according to the manufacturer’s instructions. Then, 0.5 μL of cDNA was well and incubated for 20 h. The cells that migrated through the insert’s used for quantitative polymerase chain reaction (qPCR) in a final vo- membrane were stained using 5 µM Calcein (Molecular Probes™, lume of 18 μL with SYBR Green Reaction Mix (Invitrogen) and a 0.2 μM Invitrogen) and their fluorescence was detected at an excitation of primer concentration. We performed qPCR using the Step One (Applied 480 nm and an emission of 530 nm (Tecan Infinite m200 pro). Cell Biosystems, Life technologies, Glasgow, UK) hardware and software. migration was normalized to controls and expressed as a percentage of The expression levels of target genes were normalized to internal 18 s migration. The migrated cells were photographed in an epifluorescence (rrn18s, Tataa Biocenter, Sweden) and presented as relative expressions microscope (BX61; Olympus R-FTL-T; Olympus America Inc., Center based on the 2-ΔΔCt formula. Sequences and annealing temperatures of Valley, PA, USA), using the digital image acquisition program (Olympus PCR primers are listed in Table S2. DP Controller). 2.14. Statistical analysis 2.9. Proliferation assay Experiments were performed at least in triplicate. Two-tailed Proliferation was assayed by seeding 104 cells/cm2 on 96 wells in Student’s t-tests were used for comparisons. Correlations were assessed MSC medium, and a bromodeoxyuridine assay (BrdU) was added 24 h using Kendall correlation coefficient (τ). Values of p < 0.05 were con- after cell seeding at a final concentration of 1 mM. After 2 h, DNA sidered statistically significant. The R software, (R Development Core synthesis was assayed with the Cell Proliferation ELISA BrdU kit (Roche Team, 2012) was used. All results are presented as the mean ± Molecular Biochemicals, Mannheim, Germany), according to manu- standard error (MD ± SE). facturer’s instructions. 3. Results 2.10. Small interfering mRNA transfection 3.1. ITGA6 was related to higher clonogenicity and migration but was lost To inhibit ITGA6 specifically, a small interfering mRNA (siRNA) with culture passages knockdown assay was performed using a commercial ITGA6-targeting siRNA (sc-43129, Santa Cruz, CA, USA) with an unspecific siRNA We first focused our attention on testing the intrinsic potentials of (siCTL, sc-37007, Santa Cruz, CA, USA). The transfections were per- BM-MSC from distinct donor sources for clonogenicity, migration and formed using Lipofectamine (Invitrogen) according to the manufacturer ITGA6 expression at the end of different passages during amplification instructions. Briefly, cells were seeded at a density of 15,000 cells/cm2, to determine whether ITGA6 could correlate with these potentials. incubated with 2 µM siRNA, and lysed with a western blot loading The highest ITGA6 expression was found in a-MSC and ha-MSC at buffer 72 h after transfection to evaluate protein expression. passage number (P) 1, while ed-MSC showed the lowest expression. In all cases, ITGA6 levels decreased at P5 though a-MSC and ha-MSC 2.11. Immunofluorescence and confocal microscopy shared similar expression levels and ed-MSC showed again the lowest expression (Fig. 1A). CFE assays at P1 revealed that a-MSC had the Cells grown on glass coverslips were fixed with 4% paraformalde- highest clonogenic capacity, followed by ha-MSC, whereas the ed-MSC hyde, permeabilized, and blocked. Coverslips were then incubated with showed the lowest capacity. CFE decreased with the passage number in primary antibodies for 16 h at 4 °C in a humidified chamber. After all cases (Fig. 1B). However, a-MSC continued to exhibit higher CFE washes, proper secondary antibody was added for 60 min at 37 °C in a than ha-MSC while ed-MSC showed the lowest CFE at P5. To further humidified chamber. The coverslips were mounted upside down with study the correlation between ITGA6 expression and clonogenicity, we mounting medium (Vectashield, Vector Laboratories, CA, USA) and the studied MSC from aged passages and other sources. So, we analyzed a- nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole). MSC at P9 and AT-MSC at P1 for CFE and ITGA6 expression (Fig. S1). Cells were observed in an epifluorescence microscope. For confocal Both presented insignificant CFE results that correlated with a very low microscopy with MSC spheroids, spheres were treated as described expression of ITGA6. Kendall coefficient showed a strong correlation 3 N. Nieto-Nicolau, et al. Stem Cell Research 47 (2020) 101899 Fig. 1. ITGA6 expression, clonogenic and cell migration potential of BM-MSC from different sources. (A) Expression of ITGA6 + at passage 1 (P1) and 5 (P5) and isotype negative control (Neg). (B) Colony forming efficiency (CFE) assay at P1, P5. (C) transwell migration assays using calcein staining and the percent of migrated cells at P1 and P5 (Bar = 50 μm). BM-MSC from alive healthy (a-MSC), heart arrest (ha-MSC), and encephalic death (ed-MSC) donors. Results are presented as mean ± SE from 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t-tests (*, p < 0.05; **, p < 0.01; ***, p < 0.001 P1 compared to P5; ##, p < 0.01; ###, p < 0.001 compared to a-MSC P1; &, p < 0.05 compared to a-MSC). between CFE and ITGA6 expression in all analyzed conditions MSC highly reduced their potential. The percentage values of migration (τ = 0.71; p = 0.003) (Fig. S2). in ed-MSC were again the lowest (Fig. 1C). Regarding migration, results showed similar patterns to those for CFE, decreasing with passage number. At P1, a-MSC and ha-MSC 3.2. MSC could retain mesenchymal phenotype although showed impaired showed the highest percentages of migration, without significant dif- clonogenicity or migration ferences, whereas ed-MSC group had the lowest percentage of migra- tion. Thus, a-MSC and ha-MSC reduced its migration at P5, although ha- Next, to demonstrate the maintenance of mesenchymal phenotype, 4 N. Nieto-Nicolau, et al. Stem Cell Research 47 (2020) 101899 (caption on next page) 5 N. Nieto-Nicolau, et al. Stem Cell Research 47 (2020) 101899 Fig. 2. Multi-lineage differentiation potential of BM-MSC from different sources. (A) In vitro adipogenesis differentiation at passage 1 (P1), 5 (P5) and 8–10 (P8/10) (Bar = 50 μm) and Oil Red staining elution quantification. (B) In vitro osteogenesis differentiation at P1, P5 and P8/10 (Bar = 50 μm) and Alizarin Red staining elution quantification. (C) In vitro chondrogenesis differentiation stained with Alcian Blue at P1, P5 and P8/10 (Bar = 200 μm). (D) Culture control conditions for adipogenesis and osteogenesis differentiation (Bar = 50 μm). BM-MSC from alive healthy (a-MSC), heart arrest (ha-MSC), and encephalic death (ed-MSC) donors. Results are presented as mean ± SE from 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t-tests (*, p < 0.05 treated compared to respective untreated controls in the same passage; &, p < 0.05 treated P1 compared to treated a-MSC at P1; ##, p < 0.01; ###, p < 0.001 treated P5 compared to respective treated P1; %, p < 0.05 ed-MSC P5 compared to a-MSC P5; $, p < 0.05 treated P8/10 compared to respective treated P5; ?, p < 0.5 treated P8/10 compared to treated a-MSC at P8/10). we performed trilineage differentiation and phenotypic analysis for the lower levels (Fig. 4A). At 168 h, the expression of p53, p21 and the AKT MSC that were analyzed before for ITGA6 expression, CFE and migra- pathway were not different between all the situations (Fig. 4C). Al- tion at different passages. though the DPT remained lower in the ITGA6 + fraction-derived cells, All groups (a-, ed-, ha-MSC) successfully differentiated to osteo- the DPT increased in all the situations in comparison with the DPT at genic, adipogenic, or chondrogenic lineages during amplification up to 48 h (Fig. 4D). Collectively, these results showed a reduction in the P5, although there were differences in the differentiation potential proliferation until the end of the assay. (Fig. 2). Adipogenesis and osteogenesis were higher in a-MSC, followed ITGA6 inhibited cells increased the expression cell cycle proteins by ha-MSC and ed-MSC had the lowest differentiation capacity at P1. p21, p27 and p53 (Fig. 4E). Accordingly, p-AKT was hindered, de- Moreover, adipogenesis decreased through amplification while osteo- monstrating the effect of ITGA6 on proliferation and supporting pre- genic differentiation was enhanced at P5. However, chondrogenesis and vious results (Fig. 3A). adipogenesis failed at P8/10. Further, the phenotypic characterization was maintained up to P5 in a-MSC. In the cadaveric donors, phenotypic 3.5. Fibronectin enhanced more ITGA6 expression and CFE than laminin- characterization was lost at P5 (Table S3). 111 at low densities 3.3. ITGA6 selected smaller BM-MSC with greater proliferation, Then, to assay the possible influence of substrates on ITGA6, we clonogenicity and differentiation potentials assessed the changes in ITGA6 expression, CFE and migration induced by fibronectin or laminin-111 under different confluency conditions. Then, we decided to check whether ITGA6 could be related to BM- At day 1 (D + 1), cells seeded at low confluency on fibronectin MSC potentials. So, cell sorting assays or function-blocking antibodies expressed higher ITGA6 levels than either laminin-111 or controls as were used to analyze proliferation, clonogenicity, differentiation and showed by flow cytometry (Fig. 5A and B) and immunofluorescence migration. (Fig. 5C). There was an important reduction in ITGA6 expression 7 days Sorted ITGA6 + cells showed increased proliferation compared after culture (D + 7) in all the situations. In fact, cells grown on fi- with the ITGA6 − fraction (Fig. 3A). In addition, ITGA6 + cells showed bronectin or laminin-111 showed more reduced ITGA6 expression than augmented CFE and increased osteogenic and adipogenic differentia- controls (Fig. 5A–C). tion potential (Fig. 3C–D). Moreover, ITGA6 function-blocked cells with Cells seeded on fibronectin at low confluence demonstrated the specific antibodies presented reduced migration (Fig. 3F). Besides, we highest CFE when reseeded on plastic (Fig. S3). BM-MSC grown on used flow cytometry to determine the differences in cell size of both laminin-111 and fibronectin after 7 days led lesser CFE compared with fractions, showing that ITGA6 + cells were smaller than ITGA6 − cells controls. When examining the changes in cellular migration induced by (Fig. 3B). confluence, cells had impaired migration at D + 7 compared with the cells seeded at D + 1 at lower confluence (Fig. S3). 3.4. Culture confluence regulated ITGA6 affecting proliferation 3.6. Fibronectin improved more proliferation than laminin-111 activating Next, we aimed to study the extrinsic modulation of ITGA6.To ex- AKT pathway plore the effects of culture confluence on proliferation and ITGA6 ex- pression, we purified ITGA6 + cells and ITGA6- cells by cell sorting, The influence of substrates was further studied testing cell pro- seeded them and compared them with unsorted cells. Then, we ana- liferation on both laminin-111 and fibronectin at different times (24, 48 lyzed ITGA6 expression at 48 h and 168 h post-seeding. We also eval- and 72 h) by BrdU incorporation assays and cell cycle proteins analysis. uated cell cycle proteins p53, p21 and the AKT pathway and calculated Increased proliferation was seen at 24 h after the seeding, but the the doubling population time (DPT). To further explore the relationship most enhanced proliferation was detected in cells grown on fibronectin, between ITGA6 and cell cycle proteins, we performed mechanistic followed by those seeded on laminin-111 in comparison with controls siRNA ITGA6 inhibition assays and analyzed the effects on cell cycle (Fig. 6A). Surprisingly, the lowest p-AKT expression was detected on proteins. laminin-111 substrates (Fig. 6B). There was an increase in the propor- At 48 h, almost all ITGA6 + fraction-derived cells expressed ITGA6 tion of p-AKT on fibronectin, indicating activation of the AKT pathway (Fig. 4A), while the unsorted cells expressed lessened ITGA6 expression. followed by controls. However, p21, p27, and p53 expressions were The ITGA6 + fraction-derived cells showed a very weak expression of decreased on both laminin-111 and fibronectin compared with controls. p53 and p21 cell cycle inhibitor proteins and an increased proportion of Again, the lowest p21 and p27 expressions were found on fibronectin the growth associated signal transduction pathway p-AKT (Fig. 4B). substrates, corroborating the BrdU incorporation results (Fig. 6B). This correlated with decreased doubling population times (DPT), in- dicating higher proliferation (Fig. 4D). ITGA6- fraction-derived cells 3.7. Laminin-111 was more specific for ITGA6 binding although fibronectin expressed ITGA6 although these levels were lower than in unsorted enhanced the expression of growth factors cells. ITGA6- fraction-derived cells showed the lowest AKT activation and the greatest increase of p53 and p21 (Fig. 4B), which correlated To further understand the effects of substrates, we assayed the with higher DPT, indicating lower proliferation (Fig. 4D). specificity of ITGA6 for binding laminin-111 or fibronectin and ana- The expression of ITGA6 decreased with culture time in all the si- lyzed the migration of MSC towards these substrates. Moreover, we tuations at 168 h, although ITGA6 + fraction-derived cells expressed explored the mRNA expression of growth factors that could affect cell higher levels of ITGA6, while ITGA6- fraction-derived cells expressed adhesion, proliferation, and migration. 6 N. Nieto-Nicolau, et al. Stem Cell Research 47 (2020) 101899 Fig. 3. ITGA6 expression and proliferative potential. (A) Analysis of proliferation between ITGA6 + and ITGA- sorted cells by bromodeoxyuridine (BrdU) in- corporation assay. (B) Size analysis of ITGA6 + and ITGA6- sorted cell cultures by flow cytometry. (C) Colony forming efficiency (CFE) of ITGA6 + and ITGA6- sorted cell cultures. (D) In vitro adipogenesis differentiation and Oil Red staining elution quantification of ITGA6 + and ITGA6- sorted cell cultures. (E) In vitro osteogenesis differentiation and Alizarin Red staining elution quantification of ITGA6 + and ITGA6- sorted cell cultures. (F) Percent of migrated cells using ITGA6 blocking antibody compared to control cells (IgG isotype) using through transwell migration assays. Cells stained with (Bar = 50 μm). Results are presented as mean ± SE from 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t-tests (*, p < 0.05; **, p < 0.01; ***, p < 0.001). 7 N. Nieto-Nicolau, et al. Stem Cell Research 47 (2020) 101899 (caption on next page) 8 N. Nieto-Nicolau, et al. Stem Cell Research 47 (2020) 101899 Fig. 4. ITGA6 regulation by culture confluence. (A) Sorting of both ITGA6 + and ITGA6- fractions by flow cytometry and the percent of ITGA6 of the both fractions and unsorted cells (CTL) after seeding them for 48 h and 168 h. (B) Western blot analysis and quantification for cell cycle proteins p53, p21 and AKT signaling pathway at 48 h after seeding. (C) Western blot analysis and quantification for cell cycle proteins p53, p21 and AKT signaling pathway at 168 h after seeding. (D) Doubling cell population times (DPT) of ITGA6+, ITGA6- and CTL after being seeded for 48 h and 168 h. (E) Western blot analysis for cell cycle proteins and AKT in small interference mRNA transfected cells for ITGA6 inhibition (siITGA6), compared to control cells transfected with unspecific interference mRNA (siCTL). Results are presented as mean ± SE from 3 to 4 independent experiments. Statistical analysis was performed using two-tailed Student’s t-tests (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Cell adhesion on laminin-111 decreased at every time point (10, 20, (MMP2), and interleukin 6 (IL-6) increased in cells seeded on fi- 30, and 60 min) (Fig. 6C), while adhesion on fibronectin increased at bronectin for 24 h (Fig. 6D). Only MMP2 and IL-6 expressions were 10 min and 30 min. Using an ITGA6 function-blocking antibody, cell increased in cultures grown on laminin-111 for 24 h. adhesion was lowered on the laminin-111 substrate, indicating a spe- cific participation of laminin-111 binding in MSC (Fig. 6E). Improved 3.8. Spheroid-derived MSC presented higher ITGA6 expression, migration from the apical to the basal side was observed on both the proliferation, greater p-AKT activation and improved progenitor markers laminin-111 -and fibronectin-coated inserts (Fig. 6F). expression The mRNA expressions of epidermal growth factor receptor (EGFR), transforming growth factor β (TGF-β), matrix metalloproteinase 2 Finally, we explored a strategy to increase MSC potentials. To do so, Fig. 5. ITGA6 expression on different substrates and the effects of culture confluence. (A–B) ITGA6 expression in BM-MSC by FACS on the different substrates at low (D + 1) and high (D + 7) confluence cell culture conditions. Isotype negative controls (Neg). (C) Immunofluorescence for ITGA6 staining in BM-MSC on Ctl, LN and FN at low (D + 1) and high (D + 7) confluence (Bar = 20 μm). Results are presented as mean ± SE from 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t-tests (*, p < 0.05; **, p < 0.01; ***, p < 0.001). 9 N. Nieto-Nicolau, et al. Stem Cell Research 47 (2020) 101899 Fig. 6. Implications of different substrates in BM-MSC behavior. (A) Cell proliferation assay by bromodeoxyuridine (BrdU) on laminin-111 (LN) and fibronectin (FN), and plastic (Ctl). (B) Western blot analysis for p21, p27 and p53 and AKT on LN and FN. (C) Cell adhesion assay on extracellular matrix proteins LN, FN, bovine serum albumin (BSA) and Ctl. (D) Expression of EGFR, TGF-β, MMP2 and IL-6 mRNA in BM-MSC by qPCR on LN, FN and Ctl. (E) Cell adhesion assay on LN, FN and BSA with (ITGA6 blocking) and without (IgG isotype) blockage on different substrates. (F) Percent of migrated cells trough transwells coated with LN and FN. Results are presented as mean ± SE from 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t-tests (*, p < 0.05; **, p < 0.01; ***, p < 0.001). we generated spheroids under non-adherent conditions, seeded them MSC. However, spheroid-derived MSC showed lower levels of NANOG. (spheroid-derived MSC), analyzed the expression of ITGA6 and their Increases in Sox2 and Oct4 could be also noticed in spheroid-derived capacities using as controls monolayer cultured MSC at the same pas- MSC compared with controls in representative micrographs by im- sage (Ctl). In these experiments we evaluated the migration potential, munofluorescence (Fig. 7G). the proliferation, the clonogenicity and the mRNA expression levels of SOX2, OCT4 and NANOG. Since MSC express low or negligible levels of 4. Discussion Sox2 and Oct4 (Han et al., 2014; Matic et al., 2016; Pierantozzi et al., 2011), we hypothesized that any increase in these pluripotency factors In this study, we showed that ITGA6 expression is associated to could be linked to a higher progenitor state (Boiani and Schöler, 2005; intrinsic progenitor potentials and that could be upregulated ex- Orkin et al., 2008). IPSC were used for comparison. trinsically by cell culture microenvironment, improving the prolifera- Spheroid-derived MSC expressed more ITGA6 than monolayer-de- tion, migration and clonogenicity while activated AKT pathway and rived MSC at the same passage (Fig. 7A). Moreover, the spheroid-de- decreased cell cycle inhibitor proteins. rived MSC showed increased CFE, migration potential, and improved CFE is a widely accepted assay for quantifying the number of pro- proliferation (Fig. 7B–D). Thus, the highest levels of p-AKT were pre- genitor cells in MSC cultures (Cordeiro-Spinetti et al., 2014) and for sent in MSC spheroids followed by spheroid-derived MSC (Fig. 7E). predicting the replicative potential (Digirolamo et al., 1999). BM-MSC Proliferating cell nuclear antigen (PCNA) was higher in spheroid-de- with higher CFE, migration and differentiation potentials were those rived MSC and MSC spheroids and cell cycle inhibitor protein p27 was expressing more ITGA6, and those potentials as well as ITGA6 were lost downregulated in both conditions. Moreover, a marked upregulation in during passage amplification, even if MSC retained mesenchymal phe- the mRNA expression levels of the SOX2, OCT4 and NANOG were noted notype. In agreement, we demonstrated that ITGA6 + cells had in- in MSC spheroids (Fig. 7F). MSC spheroids expressed similar levels of creased CFE, supporting previous research (Lee et al., 2009; Yu et al., SOX2 and NANOG than those expressed by IPSC, although IPSC showed 2012) and that a discrete reduction in ITGA6 expression can hinder higher expression of OCT4. Spheroid-derived MSC downregulated the CFE. Cell size and progenitor potential are related in MSC, with smaller expression levels of the pluripotency factors, though retained higher cells showing enhanced potential for differentiation and rapid self-re- expression of OCT4 and SOX2 than the controls, monolayer cultured newal (Colter et al., 2001). Our findings pointed out that ITGA6 + cells 10 N. Nieto-Nicolau, et al. Stem Cell Research 47 (2020) 101899 Fig. 7. Stemness potential, proliferation and migration of ITGA6 + spheroid-derived MSC. (A) Expression of ITGA6 in spheroid-derived MSC by FACS analysis compared to monolayer control cells (Ctl) and isotype negative controls (Neg). (B) Colony forming efficiency (CFE). (C) Transwell migration assay using calcein staining and the percent of migrated spheroid-derived MSC (Bar = 50 μm). (D) Spheroid-derived MSC proliferation capability by bromodeoxyuridine (BrdU) incorporation assay. (E) Western blot analysis p27, PCNA and AKT cell signaling pathway in MSCm/p, MSCm and (Ctl). (F) Expression of SOX2 and OCT4 mRNA in spheroid-derived MSC, spheroid and Ctl by qPCR. (G) Representative immunofluorescence micrographs for SOX2 and OCT4 in spheroid-derived MSC and Ctl (Bar = 20 μm). Results are presented as mean ± SE from 3 independent experiments. Statistical analysis was performed using two-tailed Student’s t-tests (*, p < 0.05; **, p < 0.01; ***, p < 0.001). were smaller and had an increased differentiation capacity, supporting showed that ITGA6 expression decreased as confluency increased, the greater progenitor potential of this fraction. Our data suggests that confirming the microenvironmental regulation of this integrin. So, the ITGA6 is regulated by an intrinsic program that indicates the progenitor decrease in proliferation in high confluent cultures was related to the potential of each BM-MSC. So, ITGA6 could be a candidate for pre- change in the expression of ITGA6 and the regulation of AKT pathway dicting MSC progenitor potential. Further, we confirmed that ITGA6 and cell cycle inhibitor proteins, pointing out that the ITGA6 can act as increased proliferation through AKT and cell cycle inhibitor proteins a proliferation regulator in response to culture confluence. ITGA6 was p53, p27, and p21 agreeing with previous reports in umbilical cord expressed in cultures from ITGA6-cells, showing that lower confluences blood MSC (Yu et al., 2012). can induce ITGA6 expression, further indicating the extrinsic modula- MSC seeded at low density can increase progenitor gene expression tion of ITGA6 expression by culture confluence. such as OCT4 and NANOG (Lee et al., 2013, 2009). In agreement, we Levels of ITGA6 and CFE increased on cells grown for 1 day at low 11 N. Nieto-Nicolau, et al. Stem Cell Research 47 (2020) 101899 confluence on fibronectin, though ITGA6 was more specific for laminin- original draft, Writing - review & editing. Raquel M. Torre: Data 111 binding than fibronectin, which suggests that fibronectin could curation, Investigation. Oscar Fariñas: Resources. Andrés Savio: favor progenitor potential in MSC at low densities in comparison with Resources. Anna Vilarrodona: Resources. Ricardo P. Casaroli- laminin-111. Although, further research is needed to clarify the exact Marano: Conceptualization, Methodology, Supervision, Visualization, mechanism through fibronectin could increase ITGA6 expression, there Project administration, Funding acquisition, Writing - review & editing. was an improved BM-MSC proliferation on fibronectin by AKT activa- tion and a decrease of cell cycle inhibitor proteins. More studies are Declaration of Competing Interest needed also to study the effects of other laminin isoforms, though la- minin-111 increased proliferation through impaired expressions of cell The authors declare that they have no known competing financial cycle inhibitor proteins, but not by AKT activation, which was in- interests or personal relationships that could have appeared to influ- hibited. Overall, our data suggest a bilateral interplay between AKT ence the work reported in this paper. pathway and ITGA6 expression. So, our results from ITGA6 mechanistic assays and substrate-seeding analyses indicate that AKT pathway acti- Acknowledgments vation can play a central role in ITGA6 expression, supporting previous data (Yu et al., 2012). To note, MSC treatment with TGF-β increases This work was funded in part by a grant from Fondos de ITGA6 expression (Shangguan et al., 2012), which is consistent with Investigaciones Sanitarias del Instituto Carlos III (FIS14-PI00196 and our results showing that TGF-β was only increased on fibronectin sub- FIS18-PI00355) and Fundació Marató TV3 (20120630-30-31), and co- strates where ITGA6 was augmented. Moreover, EGFR is known to financed by the European Regional Development Fund (FEDER) of the activate the AKT signaling pathway (Garay et al., 2015; Huang et al., European Union (EC). 2013; Krampera et al., 2005; Tamama et al., 2006), expression that was increased on fibronectin, where AKT signaling pathway was activated. Appendix A. Supplementary data Moreover, the highest IL-6 expression was found also in fibronectin substrates, and IL-6 acts as a growth factor for MSC which deficiency Supplementary data to this article can be found online at https:// decreases the MSC progenitors in vivo (Rodríguez et al., 2004). There- doi.org/10.1016/j.scr.2020.101899. fore, these results could provide new insights about ITGA6 regulation. More studies are needed regarding MSC migration and homing References (Ullah et al., 2019). Here, we showed that when ITGA6 decreased or was functionally inhibited, migration of BM-MSC was impaired. This is Alt, E.U., Senst, C., Murthy, S.N., Slakey, D.P., Dupin, C.L., Chaffin, A.E., Kadowitz, P.J., consistent with previous in vivo reports showing that ITGA6 blockade Izadpanah, R., 2012. Aging alters tissue resident mesenchymal stem cell properties. reduces the homing of hematopoietic progenitors to the bone marrow Stem Cell Res. 8, 215–225. https://doi.org/10.1016/j.scr.2011.11.002. 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