Molecular and phenotypic characterization of human amniotic fluid-derived cells: a morphological and proteomic approach.

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Stem Cells and Development Molecular and phenotypic characterization of human amniotic fluid-derived cells. A morphological and proteomic approach. (doi: 10.1089/scd.2014.0453) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

1 Molecular and phenotypic characterization of human amniotic fluid-derived cells. A morphological and proteomic approach.

Caterina Pipino,1,5,6* Laura Pierdomenico,2,5,6* Pamela Di Tomo,1,5,6 Fabrizio Di Giuseppe,1,5,6 Eleonora Cianci,1,5,6 Iolanda D’Alimonte,1,6 Caterina Morabito,3,5,6, Lucia Centurione,2,6 Ivana Antonucci,4,6 Maria A. Mariggiò,3,5,6 Roberta Di Pietro,2,6 Renata Ciccarelli,1,6 Marco Marchisio,2,5,6 Mario Romano,1,5,6 Stefania Angelucci,1,5,6* and Assunta Pandolfi1,5,6*

1

Department of Medical, Oral and Biotechnological Sciences; 2Department of Medicine and

Aging Science; 3Department of Neuroscience and Imaging; 4Department of Psychological Sciences Humanities and Territory, School of Medicine and Health Sciences ‘‘G. d’Annunzio’’ University Chieti-Pescara; Italy; 5Aging Research Center (Ce.S.I.), “Università G. d’Annunzio” Foundation, Chieti, Italy; 6StemTeCh Group, Chieti, Italy.

* These authors contributed equally to this work.

Corresponding author: Assunta Pandolfi, PhD ‘‘G. d’Annunzio’’ University, Chieti-Pescara, Aging Research Center, Ce.S.I., ‘‘Università Gabriele d’Annunzio’’ Foundation, Room 421, Via Luigi Polacchi, 66013 Chieti, Italy. Fax: #39-0871-541425; e-mail: [email protected]

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2 Abstract Mesenchymal Stem Cells derived from Amniotic Fluid (AFMSCs) are multipotent cells of great interest for regenerative medicine. Two predominant cell types, i.e. Epithelial-like (Elike) and Fibroblast-like (F-like), have been previously detected in the amniotic fluid (AF). In the present study, we examined the amniotic fluid from 12 donors and observed the prevalence of the E-like phenotype in 5, whereas the F-like morphology was predominant in 7 samples. These phenotypes showed slight differences in membrane markers, with higher CD90 and lower Sox2 and SSEA-4 expression in F-like than in E-like cells, whereas CD326 was expressed only in the E-like phenotype. They did not show any significant differences in osteogenic, adipogenic or chondrogenic differentiation. Proteomic analysis revealed that samples with a predominant E-like phenotype (HC1) showed a different profile than those with a predominant F-like phenotype (HC2). Twenty-five and eighteen protein spots were differentially expressed in HC1 and HC2 classes, respectively. Of these, 17 from HC1 and 4 from HC2 were identified by mass spectrometry. Protein-interaction networks (PIN) for both phenotypes showed strong interactions between specific AFMSC proteins and molecular chaperones, such as pre-proteasomes and mature proteasomes, both important for cell cycle regulation and apoptosis. Collectively, our results provide evidence that, regardless of differences in protein profiling, the prevalence of E-like or F-like cells in AF does not affect the differentiation capacity of AFMSC preparations. This may be valuable information with a view to the therapeutic use of AFMSCs.

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3 Introduction Human amniotic fluid protects the embryo development and is routinely used for diagnostic purposes. It contains cells, of fetal and/or amniotic membrane origin, which express stemness markers. Amniotic fluid cells are a heterogeneous population of epithelial origin derived from all three germ layers, its composition depending on the gestational period [1,2]. Based on morphologic, proliferation and biochemical features, these cells are classified in three main groups: epithelioid (E-like), fibroblastic (F-like) and amniotic fluid specific cells (AF-like) [3,4], the F-like being predominant [5]. A subpopulation of pluripotent stem cells can also be found in the amniotic fluid as well as multilineage mesenchymal stem cells [6,7]. It is to be noted that all these cells maintain a normal karyotype during sub-culturing in vitro and do not retain tumorigenic activity when injected into immune-compromised mice [8]. Moreover they can differentiate into the cells of the three germ layers [7]. These properties make AF-derived cells excellent candidates for cell therapy. Indeed, due to their immunemodulatory properties, AF-derived cells may be utilized in graft-versus-host disease as well as in other immune disorders [9,10]. Given the heterogeneity of cells derived from the amniotic fluid, a full characterization of their phenotype is warranted before they are introduced in therapy [11]. Previous proteomic studies on the total AFMSC population, including epithelioid, amniotic fluid specific, and fibroblastic cells, revealed 2400 spots that resulted in the identification of 432 different gene products. Among the proteins detected, 9 corresponded to epithelial cells, whereas 12 proteins were reported to be expressed in fibroblasts [12]. In 2007 Roubelakis et al. defined the proteomic map of AF-derived stem cells as compared to the map of Bone Marrow-Mesenchymal Stem Cells (BM-MSCs) [13]. In a later study based on cellular and biochemical characterization of two distinct selected subpopulations from amniotic fluid, F-like cells (Spindle Shape-AF-Mesenchymal Progenitor Cells, SS-AFMPCs) and E-like cells (Round Shape-AF-Mesenchymal Progenitor Cells, RS-AF-MPCs) [14], 25 proteins were found to be differentially expressed in SS-AF-MPCs as compared to RS-AF-MPCs, reflecting their high proliferative rate, differentiation potency, lentiviral

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4 transduction efficiency and long-term survival in vivo. These SS-AF-MPCs colonies, mechanically isolated by AF samples, can generate millions of cells with features that will be of great importance for cell and gene therapy. In agreement with Roubelakis and colleagues [13,14], we recently observed that, at very early passages in vitro, unselected AFMSC cultures contained a mixture of both round/polyhedral shaped E-like and elongated F-like cell types at almost equal frequency. By contrast, at passage 7-8 the relative abundance of these phenotypes varied [15]. Interestingly, a proteomic analysis was also recently performed on various different culture passages of CD117+ AFSCs, which exhibited variations in protein expression mainly occurring at early passages [16]. We thus conjectured that a more detailed study of unselected AFMSC cultures might be useful at medium/late passages, exploring their proteomic identity and specific properties. We preferred the mixed cell approach to separation of the two cell populations so as to minimize cell manipulation in case these cells might be used clinically. In vivo, however, AF-derived stem cells are a heterogeneous population composed of multiple categories of cells with specific morphological, biochemical and growth features, suggesting the possibility that in the amniotic fluid epithelial and stromal may coexist based on the epithelial mesenchymal transition (EMT), a molecular pathway necessary for AF epithelial cells to acquire a stromal phenotype with immunoreactivity and differentiative ability as expected of true stem cells [17]. The aim of this work supports this hypothesis, and contributes to our knowledge of the biological and biochemical aspects of AF stem cell heterogeneous populations in vitro by discovering molecular markers indicative of phenotypical and functional cell clones from stromal and epithelial cells. Furthermore, this work shows that the heterogeneity of AFderived cells could not interfere with their therapeutic potential.

Materials and Methods

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5 Isolation and culture of human amniotic fluid mesenchymal stem cells Human Amniotic Fluid (AF) samples were obtained from women undergoing prenatal diagnosis at 16-18 weeks of pregnancy after written informed consent approved by the Ethics Committee of the University of Chieti. After withdrawal, cells from AF samples were cultured in low glucose DMEM (PAA Laboratories, Dartmouth, MA, USA) supplemented with 20% fetal bovine serum (FBS, PAA), 5 ng/ml recombinant human basic FGF (R&D Systems, Minneapolis, United States) and incubated at 37°C with 5% CO2 in humidified atmosphere. The first medium change was performed after 7 days. Once the cultures had reached 7080% confluence, cells were harvested and re-plated at 3000 cells/cm2. The cells were kept in culture up to 8 passages. For all the experiments cells were taken from passage 3 [18]. Phenotype characterization was performed using 12 cell samples at the 3rd culture passage. E-like and F-like AFMSC phenotypes were spontaneously obtained along the in vitro passages. We noted that at passages 7-8 the E-like AFMSC subtype was characterized by the presence of  70% of round/polyhedral cells, and the F-like AFMSC by the occurrence of  70% elongated/spindle-shaped cells. We studied 12 cell samples and observed the prevalence of the E-like phenotype in 5, whereas F-like morphology was predominant in 7 samples. Phenotype characterization of both populations was performed at passage 7-8 (see Table 2). To obtain enough cells to further characterize the molecular and biochemical features of AFMSCs by proteomic analysis, we employed 7 cell samples from both populations up to the 8th passage.

Phenotyping Cell Staining for Flow Cytometry analysis Twelve cell samples were stained for surface or intracellular antigens, as previously described [19,20]. Briefly, 5x105 cells/sample were incubated with 100 μl of 20 mM ethylenediaminetetraacetic acid (EDTA) at 37°C for 10 min and washed. Washing buffer (1X

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6 PBS, 0.1 % sodium azide and 0.5 % bovine serum albumin, BSA) was used for all washing steps (3 ml of washing buffer and centrifugation 8 min at 4˚C at 400 x g). Staining of surface antigens Samples were suspended in 100 μl washing buffer containing the appropriate amount of surface antibody (Table S1) and then incubated for 30 minutes at 4˚C in the dark. Tubes were washed and cells were suspended with 1 ml 0.5% paraformaldehyde, then incubated for 5 min at RT. Staining of intracellular antigens Cells were suspended in 1 ml of Perm 2 (BD) added to each tube and incubated at room temperature in the dark for 10 min. Then the procedure was the same as described for surface antigens, using the appropriate amount of intracellular antibody (Table S1). Flow cytometry measurement Cells were fixed with 1 ml 0.5% paraformaldehyde and stored at 4˚C in the dark until acquisition. Finally, they were analyzed on a FACSCanto flow cytometer (BD), using Diva™ software (BD). Quality control included regular check-ups with Rainbow Calibration Particles (BD Biosciences). Debris was excluded from the analysis by gating on morphological parameters; 20,000 non-debris events in the morphological gate were recorded for each sample. To assess non-specific fluorescence we used specific irrelevant controls. All antibodies were titrated under assay conditions and optimal photomultiplier (PMT) gains were established for each channel [21]. Data were analyzed using FlowJo™ software (TreeStar, Ashland, OR). The Mean Fluorescence Intensity Ratio (MFI Ratio) was calculated by dividing the MFI of positive events by the MFI of negative events [22].

Real-time PCR Total RNA from AFMSCs cultured in basal and osteogenic/adipogenic medium was isolated using the RNeasy Plus Universal Mini Kit (Qiagen Inc., Valencia, CA) according to manufacturer’s instructions. The quality of total RNA was assessed by measuring the A260/280 ratio using a spectrophotometer. For the reverse transcriptase reaction, M-MLV

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7 Reverse Transcriptase reagents (Sigma-Aldrich, St. Louis, MO, USA) were used. Real-Time RT-PCR was carried out with the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Expression of Alkaline Phosphatase (ALP), Runt-related transcription factor 2 (RUNX2) and Osteopontin (OPN) was evaluated at 3, 7 and 14 days in epithelial- and fibroblast- like AFMSCs cultured in osteogenic medium at passage 3. Commercially available TaqMan Gene Expression Assays (RUNX2, Hs00231692_m1, ALP, Hs01029144_m1, OPN Hs00959010_m1) and the TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) were used according to standard protocols. For adipogenic

differentiation,

expression

of

the

PPARγ

(Hs01115513_m1),

FABP4

(Hs01086177_m1) and LPL (Hs01012567) gene was evaluated up to day 10. In both quantitative PCR, Beta-2 microglobulin (B2M, Hs99999907_m1, Applied Biosystems, Foster City, CA, USA) was used for template normalization and duplicates were set up for each sample. P values 70%) E-like or F-like phenotype, with a view to determining the impact of the predominant phenotype on various functional and structural parameters. The mixed cell approach was preferred to separation of the two cell populations as reported by Roubelakis et al. (2011) so as to minimize cell manipulation in case these cells should be used clinically.

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17 The two phenotypes differentiated equally well into osteogenic, adipogenic and chondrogenic lineages (Fig. 3). This is an intriguing observation, in apparent contrast with previous data showing differences in osteogenic and adipogenic differentiation capacity of the two phenotypes [14]. One likely explanation is that cross-talk between the two phenotypes may stabilize functions of either cell type, thus maintaining homogeneous differentiation properties regardless of predominance or relative abundance. To gain a broader view of the phenotypic profile of AFMSC, we carried out proteomic and protein-protein network analyses (Figs. 4 and 5). Interestingly, the E-like and F-like phenotypes shared ∼ 200 proteins with ∼ 43% of the total proteome being represented by cytoskeletal proteins and by components of the protein biosynthesis machinery. Unlike what we had observed in Wharton’s jelly mesenchymal stem cells [43], the metabolism enzymes mainly involved in energy catabolic processes were the second most abundant class. In addition, AFMSCs expressed several isoforms of vinculin, which regulates cell morphology during lamellipodia formation [44,45] and cell mechanics [46]. They also expressed Cofilin-1, a differentiation marker related to in vitro expansion [12], as well as a large number of proteins related to proliferation and cell homeostasis, like ubiquitin-1. In agreement with earlier data [14], Galectin 1 (GAL1) and Transgelin (TAGLN), which regulate differentiation in other stem cell types, were abundant in our AFMSCs. Moreover, a high amount of Vimentin (VIM), a marker of mesenchymal stem cells, was found in the AFMSC proteome [44]. Antioxidant proteins such as thioredoxin, peroredoxin and glutathione transferases were also present in these cells. Overall, this profile is consistent with cells with good plasticity, proliferative capability and resistance to oxidative stress. Heuristic cluster analysis suggested the total AFMSC population contained two main phenotype classes, which we termed HC1 (E-like phenotype) and HC2 (F-like phenotype). Approximately 21 proteins were differentially expressed in these classes. For instance, K2C8 , K1C18 and K1C19 were significantly higher expressed in E-like cells (Fig. 4), confirming a previous observation with purified populations [14]. Since K2C8 and K1C18 are considered markers of a more undifferentiated phenotype and K1C19 is expressed in incompletely

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18 differentiated cells [47], the increased K2C8, K1C18 and K1C19 expression in the HC1 class is consistent with a higher degree of stemness. This is also supported by the higher ITA V expression in HC1, as ITA V may be functionally involved in the maintenance of a highly migratory, mesenchymal phenotype as well as the acquisition of a stem phenotype [48]. Consistent with this, E-like cells express CD326, which is not detected in F-like preparations. Other proteins exclusively expressed in the HC1 class are related to the control of protein biosynthesis, such as an isoform of KAP 0, a major component of PKA, which regulates posttranscriptional mitochondrial gene expression [49] and is involved in several intracellular pathways including gene transcription, ion transport, metabolism, cell division and differentiation [50]. Among the proteins more abundantly expressed in class HC2, we found isoform 2 of cellular retinoic acid binding proteins (RABP2), also termed CRABP II (Table 3B) and belonging to a family of small cytosolic lipid-binding proteins, which are highly conserved during evolution [51,52]. HSPB1 was also overexpressed in HC2 cells. This protein is closely associated with the regulation of actin polymerization. The analysis of PINs from both cell phenotypes revealed a remarkable functional link: proteins involved in the regulation of cell integrity (ACTB, PFN1, HSPB1, MFAP1, KRT19, KRT8, KRT18) and cytoskeleton remodelling (cell structure and motility) are associated with the proteasome 26S complex (PSMC1,5,6; PSMD3,6,7,10,11,12,13,14; RAD23A), which modulates protein biosynthesis, folding and degradation (Fig. 5A, B), suggesting an additional role of proteasomes from the major cell proteolitic pathway, the so-called ubiquitine-proteasome pathway (UPP) [53]. Unlike HC1, the HC2 PIN showed a potential synergism between UPP and antioxidant molecular chaperons i.e. HSPs (HSPB1, HSPB6). This may prevent abnormal protein synthesis, promoting proper repair and adequate proteostasis [53,54]. The HC2 PIN also revealed multiple associations with ENOA1. This ties up with the involvement of ubiquitination events in key signalling pathways, such as innate immunity and inflammation [55].

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19 To conclude, in the present work we provide a biological and molecular characterization of the main phenotypic subpopulations in mixed AFMSC cultures in vitro taking advantage of proteomic inventories created by a 2DE comparative approach. This approach confirmed the cellular heterogeneity of the amniotic fluid and provided evidence of separate interactome networks for the E-like and F-like populations. The pathophysiological and clinical relevance of such differences as well as whether these populations have different origins or represent stages of phenotype transition remains to be determined.

Acknowledgments This study was partially supported by CARICHIETI Foundation. We thank MA Centurione for technical support with microscopy analysis.

Author Disclosure Statement No competing financial interests exist.

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Figure legends

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FIG. 1. Osteogenic, adipogenic and chondrogenic differentiation of AFMSCs. (A) Quantitative RT-PCR analysis of the specific osteogenic markers ALP, RUNX-2 and OPN shows increased levels in differentiated AFMSCs compared to undifferentiated cells. *p

Mitohormesis in muscle cells: a morphological, molecular, and proteomic approach.

Morphological and Molecular Characterization of Human Dermal Lymphatic Collectors.

Isolation and morphological characterization of ovine amniotic fluid mesenchymal stem cells.

Phenotypic Characterization Analysis of Human Hepatocarcinoma by Urine Metabolomics Approach.

Isolation, Characterization, Cryopreservation of Human Amniotic Stem Cells and Differentiation to Osteogenic and Adipogenic Cells.

Characterization of amniotic stem cells.

Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells.

Molecular characterization and in vitro differentiation of feline progenitor-like amniotic epithelial cells.

A proteomic approach to investigate the effects of cadmium and lead on human primary renal cells.

Phenotypic and molecular characterization of hyperpigmented group B Streptococci.

Characterization of Meniscal Pathology Using Molecular and Proteomic Analyses.

Molecular and phenotypic characterization of atypical Williams-Beuren syndrome.

Molecular Proteomic Characterization of a Pediatric Medulloblastoma Xenograft.

Proteomic Characterization Reveals a Molecular Portrait of Nasopharyngeal Carcinoma Differentiation.

Isolation and partial characterization of human amniotic epithelial cells: the effect of trypsin.

Isolation, primary culture, morphological and molecular characterization of circulating tumor cells in gynecological cancers.

Molecular and proteomic insight into Notch1 characterization in hepatocellular carcinoma.

Activity and characterization of a low molecular fraction present in human amniotic fluid with broad spectrum antibacterial activity.

Genotypic, phenotypic, and proteomic characterization of Candida glabrata during sequential fluconazole exposure.

Isolation and Characterization of Human Mesenchymal Stem Cells Derived from Human Umbilical Cord Wharton's Jelly and Amniotic Membrane.

Identification and characterization of angiogenesis targets through proteomic profiling of endothelial cells in human cancer tissues.

Amniotic fluid fibronectin. Characterization and synthesis by cells in culture.

Phenotypic and molecular biological analysis of human butyrylcholinesterase variants.

Phenotypic characterization of human intermediate monocytes.