Peculiar Characteristics of Human Mesenchymal Stem Cell Clones Suitable as Tissue Engineering Models

In the last few years’ stem cell research contributed to gain a fundamental understanding of how organisms grow and develop and how tissues are maintained throughout adult life. Mesenchymal stem cells (MSC) are selfrenewing, multipotent cells that are present in many adult tissues, such as bone marrow, adipose tissue, trabecular bone and muscle. More recently they have been found also in skin, liver and other tissues. Dermal skin-derived fibroblasts exhibit mesenchymal surface antigen immunophenotype and differentiation capabilities versus the three main mesenchymal tissues (bone, fat and cartilage). Hereditary Hemorrhagic Telangiectasia affects 1 in 5000 people and leads to abnormal blood vessel formation in skin and mucous membranes. We isolated human dermal fibroblasts from patients with Hereditary Hemorrhagic Telangiectasia (HHT) and healthy controls. In order to evaluate future applications of these cells in tissue engineering we compared mesenchymal properties (self-renewal, differentiation potential) of human gingival fibroblasts isolated from healthy and HHT-affected subjects using a combination of phenotypic (flow cytometry), morphologic (senescence), and functional (in vitro differentiation, colony forming unit assay and proliferation assay) criteria. Our results suggest that HHT cells were ideal candidates for tissue engineering. Journal of Tissue Science & Engineering J o u r n a l o f T iss ue S cience &ngine e r i n g


Introduction
Mesenchymal stem cells (MSCs) possess fibroblastic-like morphology, limited but long-term viability, self renewal capability and multilineage potential [1][2][3][4][5]. MSCs originate from fetus mesoderm layer and in the adult reside in a variety of tissue such as: bone marrow stem cells (BMSCs), dermal stem cells, hepatic stem cells, limbal stem cells, adipose derived stem cells and orofacial tissue (adult tooth pulp tissue, periodontal ligament and adult human dental pulp [6][7][8]; these findings indicate that also adult mammalian mucosa contains tissue derivedstem cells, and that even these fibroblastic MSCs are more plastic than previously appreciated. Several authors could demonstrate by clonal analysis that human dermis-derived fibroblasts have multipotent differentiation potential [9]. Fibroblastic mesenchymal stem-cell-like (FmSCs) present a surface marker expression pattern similar to MSCs, examination of cell size and granularity, as shown by FACS analyses, indicates the homogeneity of the FmSCs population [10].
Oral gingiva which is often resected during general dental treatments and treated as biomedical waste, is an easily obtainable tissue, and cells can be isolated from patients with minimal discomfort [11]. Interestingly, clinical observations and experimental studies consistently indicate that wound healing in the oral mucosa has better outcomes than in the skin, although the healing process and sequence are similar. The oral mucosa is composed of a thin keratinocyte layer with underlying connective tissue, the mayor constituent of this tissue are Gingival fibroblasts (GFs), that are different from skin fibroblasts [12]. These cells adhere and spread well on culture plates, and proliferate without requiring specific culture conditions.
HHT is an autosomal dominant disease characterized by diffuse visceral and muco-cutaneous telangiectases; a person with HHT has a tendency to form blood vessels that lack the capillaries between an artery and a vein; a typical HHT patient has epistaxis, mucocutaneous telangiectases and gastrointestinal bleeding in later life, even though this is only one of the possible scenarios associated with HHT disease [13][14]. This disease is caused either by mutations in endoglin on chromosome 9 (ENG; HHT1) or those of ACVRL1/ALK1 on chromosome 12 (HHT2), respectively [15]. The prevalence of this disease is on average between 10-20/100000, although it is higher in some regions [16][17][18]. Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome is caused by mutations in the gene SMAD4 (HHTJP) on chromosome 18; further genes are predicted at loci identified by linkage analyses on chromosomes 5 (HHT3) and 7 (HHT4) [19]. All these genes encode for proteins that are found in the lining of the blood vessels.
We isolated human gingival fibroblasts from biopsies of people suffering from Hereditary Hemorrhagic Telangiectasia (HHT) and from healthy controls. potential of fibroblasts derived from HHT patients in comparison to the ones from normal subjects for possible applications in tissue engineering.

Cell isolation and collection
Biopsies of oral gingiva, from adult patients (6 HHT and 2 healthy controls), were taken during surgical operation after formal consent. To establish cell cultures, tissues were minced with a scalpel without any enzymatic digestion. Fragments were then plated in a 6 cm culture dish, in growth medium composed by DMEM (Dulbecco's Modified Eagle's medium) supplemented with 10% FCS (Fetal Calf Serum), 1% Hepes, 1% Sodium Pyruvate, 1% Antibiotics (Penicilline/Streptomicine) and 1% L-glutamine (all from EuroClone ® ). Cells were kept in culture in 5% CO 2 at 37°C for one week, then fragments were manually removed and cells were allowed to expand up to 80-90% confluence.

Colony-forming unit assays (CFA)
Cells of both origins (HHT and control) were plated at 1, 2 or 3 cells/well in a 96 multiwell plate in growth medium, and cultured for 14 days, without medium changing. At the end of the culture period, cells were stained with Wright's staining and CFUs were quantified by counting colonies of >50 cells.

xCELLigence RT-CES system (Roche TM)
The RT-CES system is a real time cell analyzer developed by Roche, it is composed by three parts: an electronic sensor analyzer, a device station, and a 96-well e-plate.The e-plate containing tissue culture well is a standard flat-bottom 96-well culture plate, but in the well bottom is incorporated an in circle-on-line sensor electrode arrays. The device station, which is connected with e-plates, is placed in the incubator and connected to the electronic sensor analyzer through electrical cables. The electronic impedance of sensors electrodes is mesured to allow monitoring of changes of cells on the electrodes, this mesure gives back a parameter called cell index. The Cell Index is calculated by the dedicated software as the difference between the mesure of impedance at T0 (media alone) and the one mesured at each time point. As stated by Roche CI is a dimensionless value that is correlated to cell number and/or viability [20][21]. Under the control of the RT-CES software, experiment data are measured automatically by the sensor analyzer. For this experiment, for each cell type (in dodecuplicate), we seeded 5000 cells/cm 2 (980 cells/well) in 180 μl of DMEM 10% FCS in E-Plate 96. Cells proliferation was monitored for 1 week, with an impedance measurement every minute. This starting density is the optimal for human mesenchymal cells growth (and differentiation).

β-galactosidase staining for senescence
HHT cells at passage 31 (P31) and control cells at passage 27 (P27) were plated at density of 3500 cells/cm 2 in growth medium (DMEM 10% FCS); after 10 days of culture cells were fixed in 4% buffered paraformaldehyde. Cells were then stained with the staining solution (4.2 mM citric acid, 12.5 mM sodium-phosphate, 158 mM sodium chloride, 0.21 mM magnesium chloride, 2.21 mg/mL potassium ferrocyanide, 1.68 mg/mL potassium ferricyanide, 1 mg/mL X-Gal, pH 6.0) for 24 h at room temperature as reported by Dimri et al. β-galactosidase activity (blue cells) at pH 6 is present only in senescent cells and is not found in presenescent, quiescent or immortal cells. [22,23] Detection of telomerase activity TRAPEZE ® Gel Based Telomerase Detection kit was used to detect and evaluate telomerase activity in HHT and in control cells. This assay is a highly sensitive in vitro assay system for detecting telomerase activity and is based on improved version of the original method described by KIM et al. [24,25]. The assay is a one-buffer, two-enzyme, system utilizing PCR to enhance the sensitivity of telomerase detection in small samples. Cells were lysed and extracted DNA underwent to PCR using specific primers for telomere sequences (provided in the TRAPEZE Kit). For visualization of PCR products we run 25 µl of each sample on a 12.5% non denaturing-PAGE in 0.5X TBE buffer. This kit also provide, as positive control, a lysate of the tumoral cell line HeLa, because tumoral cells present an high telomerase activity.

Differentiation in vitro
5×10 3 cells/cm 2 were seeded in 6 well culture plates. Each culture plate was kept in growth medium until confluence, and then the medium was replaced with specific medium according to the conditions described below to induce osteogenic or adipogenic differentiation. Media were replaced once a week.
For osteogenic differentiation cells were cultured for 3 and 4 week with differentiative medium composed by DMEM 10% FCS supplemented with: 3.5mM β-Glycerophosphate and Ascorbic Acid used at a final concentrations of 50µg/ml (all from Sigma-Aldrich, Italy).
For quantification of matrix mineralization, controls and HHT cells were stained with alizarin Red-S as described previously [27]. After staining, bound dye was solubilized in 10 mM sodium phosphate (pH 7.0) containing 10% cetylpyridinium chloride and quantitated spectrophotometrically at 562 nm, using Nanodrop (Nanodrop ™ Technologies, Wilmington, USA). This quantitation methods is comparable, in accuracy, with the quantitation of hydroxyapatite by binding assay [28].

Real-Time PCR (RT-PCR)
At the end of the culture period (3 weeks for adipogenic differentiations and 40 days for osteogenic differentiations) we analyzed the expression of human genes involved in adipogenic (ADPF, SREBF1, LEP and PPAR-γ) [29] and osteogenic (ALP, COL I, ON and IBSP) differentiation [30]. Total RNA was extracted using Pure Link

Statistical analysis
Fluorescence activated cell sorting (FACS) analyses, PCR and differentiation assays were performed and analyzed in three independent experiments. Results are expressed as the mean and standard deviation. Differences between HHT and control cells were evaluated using t-test; statistical significance was established at p<0.05. Representative figures are given for stainings.

HHT gingival fibroblasts show higher colony forming capability than control cells
When plated at 1 cell/well HHT cells showed a colony forming efficiency of 97.4%, at 2 cells/well of 96.9% and of 96.3% when plated at 3 cells/well. Control cells showed a colony forming efficiency of 73.4% at 1 cell/well, at 2 cells/well of 25.8% and 28.9% for 3 cells/well Figure  1. Interestingly, we could detect a significantly higher colony forming capability in HHT cells suggesting a higher proliferation rate of HHT cells with respect to control cells. T.

HHT gingival derived fibroblasts show higher proliferation rate than healthy patients' derived cells
To test the possibility that HHT cells possess higher proliferation rate than control cells we used the xCELLigence system. The system permits the real time analysis of cell growth and attachment on the basis of mesurament of electronic impedance in each well of the plate. The impedance due to ionic strength of media alone is measured at the beginning of the experiment (T0); the presence of cells alters the impedance because the cells act as an insulator when they attach to the well's bottom. From impedance measurements instrument gives back a Cell Index, which is a relative value indicating how many cells are present in each well. For these types of experiments each measure is done in sample and control cells, thus providing a relative measure and avoiding a standard curve experiment.

Control cells show higher senescence than HHT cells
Senescence is the phenomenon by which normal diploid cells lose their ability to divide. To identify senescent cells we performed the β-galactosidase staining for senescence. β-galactosidase associated senescence is microscopically revealed by the presence of a blue, insoluble precipitate within the senescent cell after staining with x-gal. Control cells at passage 27 show a typical blue staining and also an enlarged morphology, Figure 3 B while HHT cells do not appear stained even after 31 passages Figure 3 A.

HHT higher proliferation rate is not associated with tumoral phenotype
To verify if the higher proliferation rate of HHT cells could be associated with a tumoral phenotype we compared telomerase activity of HHT and control cells using the TRAPeze Telomerase Detection Kit. No PCR amplified products should be visible in the inactivated samples (heat treated), except the 36 bp internal standard control band, while a ladder of PCR products of different weight should be visible in telomerase positive samples (Figure 4). HHT cells show low telomerase activity if compared to positive control cells (HeLa tumoral cell line) and an analogous telomerase activity if compared with cells derived from healthy persons.   Gene expression evaluation by Real time PCR of the genes involved in adipogenic differentiation (ADPF, SREBF1, LEP and PPAR-γ), is reported in Figure 6. qPCR results confirm that HHT cells differentiate along the adipogenic lineage after 3 weeks of culture in adipogenic medium, while control cells show expression pattern of undifferentiated cells. In fact there is an up-regulation of ADPF and PPARG-γ genes, key factors of adipogenic differentiation; also the expressions of SREBF1 that is involved in sterols synthesis, and leptin that is secreted by white adipocytes are upregulated respect to control. In particular there is a statistically significant up regulation of genes involved in active bone deposition (ALP) and in matrix mineralization (IBSP), and an over expression of collagen type I and osteocalcin, that are involved in late osteogenesis.

Discussion
One of the main problems of tissue engineering is the small number of cells and the excessive length of time required to regenerate tissue. For these reasons, researcher try to amplify the number of cells, find new sources of stem cells and reduce the differentiation period needed.
Stem cell therapy in HHT patients has not yet been attempted, moreover there is a paucity of information on HHT bone marrow mesenchymal stem cells due to the difficulty of obtaining them from already severely compromised people.
Therefore with this work we evaluated the mesenchymal potential of skin fibroblasts obtained from patients suffering from hereditary hemorrhagic telangiectasia (HHT) comparing them with those derived from not affected patients for further applications in tissue engineering.
As first step of the characterization we performed FACS analyses that revealed that HHT and control cells possess the same surface markers expression of mesenchymal stem cells. Given this fact we performed a set of experiments trying to assess if there were differences in their "mesenchymal" properties. Firstly we tested their self-renewal potential and the clone forming capability. Interestingly HHT clones showed, respect to the control ones, a higher clonal capability Figure  1 and also a major self-renewal potential as shown by the senescence test Figure 3. Moreover their rate of growth turned out to be higher than the one of control cells as assessed in 1 week experiments performed with the xCELLigence system by Roche Figure 2. Analysis of telomerase activity showed that HHT cells possess low telomerase activity if compared to positive control cells (HeLa tumoral cells) and an analogous telomerase activity if compared with cells derived from healthy persons.
Lastly we determined if HHT and control cells possessed mesenchymal differentiation potential, culturing them under    Figure 7 A B C. Using a combination of phenotypic (flow cytometry), morphologic (senescence), and functional (colony forming unit assay, proliferation assay and differentiation) criteria, this work could demonstrate that HHT cells fulfils the main characteristics of MSCs [31]. These cells express homogenously all MSC-related surface antigens whereas the expression of CD-proteins typical for hematopoietic cells remained undetectable, they differentiate along the adipogenic and osteogenic cell lineages, furthermore they are highly clonogenic (colony forming efficiency about 97%), show a high proliferation potential, and an enhanced capacity of self renewal along with a major stability within passages even when cryopreserved or sub cultured.
HHT cells can be isolated easily, their expansion in culture is very convenient (in vitro expansion without growth factor), thus making them ideal candidates for tissue engineering. HHT cells also represent a dynamic system suitable to the identification of new molecular targets and the development of novel drugs, which can be tested in vitro for safety or to predict or anticipate potential toxicity in humans.