Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming

Abstract Induced pluripotent stem cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell‐like state. Notwithstanding their autologous origin and their potential to differentiate towards cells of all three germ layers, iPSC reprogramming is still affected by low efficiency. As dermal fibroblast is the most used human cell for reprogramming, we hypothesize that the variability in reprogramming is, at least partially, because of the skin fibroblasts used. Human dermal fibroblasts harvested from five different anatomical sites (neck, breast, arm, abdomen and thigh) were cultured and their morphology, proliferation, apoptotic rate, ability to migrate, expression of mesenchymal or epithelial markers, differentiation potential and production of growth factors were evaluated in vitro. Additionally, gene expression analysis was performed by real‐time PCR including genes typically expressed by mesenchymal cells. Finally, fibroblasts isolated from different anatomic sites were reprogrammed to iPSCs by integration‐free method. Intriguingly, while the morphology of fibroblasts derived from different anatomic sites differed only slightly, other features, known to affect cell reprogramming, varied greatly and in accordance with anatomic site of origin. Accordingly, difference also emerged in fibroblasts readiness to respond to reprogramming and ability to form colonies. Therefore, as fibroblasts derived from different anatomic sites preserve positional memory, it is of great importance to accurately evaluate and select dermal fibroblast population prior to induce reprogramming.


| INTRODUC TI ON
Induced pluripotent stem cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell (ESC)-like state.
Since pioneering works that led to the successful reprogramming of mouse 1 and human 2,3 fibroblasts, iPSCs have been obtained from somatic cells of several other species. [4][5][6] Remarkable similarity of iPSCs to ESC, along with their origin from adult somatic cells, make iPSCs a tremendously valuable tool for regenerative medicine, disease modelling, drug discovery and testing, [7][8][9] while avoiding the ethical concerns associated with ESC.
Notwithstanding iPSCs functional resemblance to ESC, their clinical application is still prevented by severe technical problems, mostly related to both reprogramming technology and low efficiency of reprogramming. Although reprogramming technology has been significantly improved by integration-free methods based on episomal vectors, 10 synthetic modified mRNA 11 or direct delivery of reprogramming proteins, 12 efficiency of reprogramming of human cells is still as low as 2% with integration-free methods, and 6.2% at best with integration methods. 13 Several enhancers and barriers of reprogramming have been described thus far. Accordingly, novel strategies to activate enhancers or inhibit barriers are emerging. 14 Recently, reprogramming efficiencies of 80%-100% were achieved by genetic combinatorial modulation of specific signalling pathways. 15 These results shed light on the mechanisms governing cell reprogramming, but do not allow transcending the limitations to iPSC clinical translation. Additionally, evidence supporting variability in efficiency of reprogramming and in properties of iPSCs in accordance with the cell type from which iPSCs were generated was also reported. 16,17 Adult dermal fibroblast has been the first human cell successfully reprogrammed to iPSCs, 3 and, to date, it is still the most used human cell for reprogramming. Even though the potential of other adult somatic cells has been examined, fibroblasts are still the most suitable cell source to generate iPSC. Intriguingly, peripheral blood cells (PBCs) and urine-derived cells (HUCs) were emerging as alternate candidates for easiness of harvesting, but the very low efficiency of PBCs reprogramming and the very high inter-individual variation in HUCs number excreted with urine, 13 strengthened fibroblast role in human iPSCs production. Undoubtedly, dermal fibroblasts are easily accessible and propagated in culture with a single skin punch biopsy. However, according to gene expression profile analysis independently performed by different groups comparing fibroblasts from different anatomic sites, embryonic spatial organisation of fibroblast differentiation and positional memory are partially retained in adult fibroblast. 18,19 Furthermore, recently reported phenotypic and functional diversity of dermal fibroblasts 20 might need to be considered when planning reprogramming of dermal fibroblasts to iPSCs.
On this basis, we hypothesize that dermal fibroblasts differ not only in their gene expression profile, but also in other biological characteristics that might be, even partially, responsible for different response to reprogramming technology. To test our hypothesis we compared the morphology, expression of specific markers, production of soluble factors, proneness to apoptosis, proliferation rate, ability to migrate and differentiation potential of adult human dermal fibroblasts isolated from different anatomic sites and analysed any difference occurring among cell populations.

| Tissue samples
Skin fragments from five different anatomic sites (n = 25, five necks, five breasts, five arms, five abdomens and five thighs) of patients (n = 25, mean age 41.04 ± 7.624, all female patients) undergoing plastic surgery were harvested. Patients provided written, informed consent and specimens were collected, without patient identifiers, following protocols approved by the University Hospital Federico II and in conformity with the principles outlined in the Declaration of Helsinki.

| Cell culture
Samples were minced and fragments of about 2 × 1 mm, length by width, were placed under sterile coverglasses in 35 mm culture plates and cultured in DMEM (Sigma-Aldrich, St. Louis, MO) with 10% FBS (Fetal Bovine Serum) (Sigma-Aldrich) and 0.5% Penicillin-Streptomycin (Sigma-Aldrich), at 37°C in 5% CO 2 . Plates were checked daily at an inverted phase-contrast microscope (Olympus, Tokyo, Japan), and medium was replaced every 3 days. Outgrowth of cells was documented by digital image acquisition (Olympus).
Confluent fibroblasts from all regions were synchronised by being placed in 0.1% serum for 48 hours before being trypsinized and plated in the presence of 10% serum, as previously described. 21 In order to avoid any effect because of the native environment, all primary fibroblasts were cultured under the same condition in vitro for five passages. 18 Then, passage 5 fibroblasts from different anatomic sites were cultured for 1 week to evaluate their features and behaviour in vitro. All experiments were performed in triplicate.

| Scratch wound assay
1.6 × 10 5 cells/35 mm dish were plated to create a confluent monolayer. Dishes were cultured for approximately 48 hours at 37°C to allow cells to adhere and spread. Cell monolayer was scratched in a straight line with a p10 sterile pipette tip. Fresh medium was pipetted in the dish, after one wash with medium to remove debris. Culture plates were then placed under a phase-contrast microscope (Nikon, Tokyo, Japan) equipped with stage incubator (Okolab, Pozzuoli, Italy), and the migration of fibroblasts at both edges of the wound was documented acquiring one picture every 10 minutes for 12 hours by digital camera (Nikon). Data were analysed by NIS Elements software (Nikon) and expressed as mean speed of migration ± SE.

| Apoptotic index
To determine apoptotic index of fibroblast from all regions,

| Gene expression profile analysis
Culture dishes(5 × 10 5 cells/60 mm) were plated and cultured for 7 days, then processed for real-time PCR analysis as previously described. 22 Total RNA was extracted in Isol-RNA Lysis Reagent (5Prime, Hamburg, Germany), dissolved in RNase-free water and its final concentration determined at the NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA). RNA from each sample was reverse transcribed into cDNA with QuantiTect Reverse Trascription Kit (Qiagen, Hilden, Germany) and gene expression was quantified by real-time qPCR using PrecisionPLUS qPCR Master Mix (Primer Design, Southampton, UK). The primer assays for genes typical for mesenchymal cells are included in Table 1. All samples were tested in triplicate with the housekeeping gene (GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase) to correct for variations in RNA quality and quantity. Melt curve analysis was performed to assess uniformity of product formation, primer dimer formation and amplification of non-specific products. Comparative quantification of target genes expression in the samples was performed based on cycle threshold (Ct) and using the ΔΔCt method. Numbers were averaged and expressed as mean ± SE.

| Growth factor array
Dermal fibroblasts from all five regions (neck, breast, arm, abdomen and thigh) were plated at medium density (8 × 10 4 cells/35 mm) and cultured in DMEM with 10% FBS. When cells reached confluence, serum-free FibroGRO medium (Millipore, Burlington, MA) was used instead of DMEM for the next 3 days. Culture medium was then collected from fibroblasts of all dermal regions and assayed in the Human Growth Factor Array C1 (Raybiotech, Norcross, GA) to simultaneously detect 41 targets. The procedure was performed in strict accordance with manufacturer's directions. Briefly, array membranes were blocked with blocking buffer for 30 minutes at room temperature, and then 1 mL of culture medium was added to each membrane and incubated at room temperature for 2.5 hours. Membranes were then washed three times in wash buffer I and twice in wash buffer II, then incubated with biotin-conjugated antibody overnight at 4°C.
After further washes, membranes were incubated for 2 hours at room temperature with horseradish peroxidase (HRP)-conjugated streptavidin and washed one last time to remove unbound reagents.
All incubation steps were performed with agitation on orbital shaker.
Membranes were then developed with the detection buffer, exposed to film and processed by autoradiography. Numerical comparison of the signal densities of growth factors known to influence or modulate TA B L E 1 Primers of genes analysed by real-time PCR

Gene symbol
Forward sequence Reverse sequence

Amplicon length (nt)
reprogramming was performed as previously described. 23 Briefly, spot signal densities from the scanned images of arrays were obtained using ImageJ densitometry software (https://imagej.nih.gov/ij/download.html). The background was then subtracted from the densitometry data, and the obtained values were normalised to the positive control signals. Data were expressed as the mean ± SE.

| Reprogramming
Dermal fibroblasts from each anatomic site at passage 5 (n = 4) were

| Statistical analysis
Data were analysed by GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA), using one-way ANOVA test and Tukey's post test. A value of P ≤ 0.05 identified any statistically significant difference.

| Fibroblast morphology in vitro
Outgrowth of fibroblasts occurred after 6-7 days of culture ( Figure 1A).
Cells adhered to plastic culture dishes, where they spread and, in a time ranging from 14 to 21 days, they reached confluence ( Figure 1B).
Although most of the cells had spindle-shaped morphology, starshaped fibroblasts were observed in all culture dishes ( Figure 1A).

| Proliferation and apoptotic indices, ability to migrate
To avoid any effect due to native environment, all primary fibroblasts were cultured under the same condition in vitro for five passages.   Figure 2E-G).

| Expression of mesenchymal and epithelial markers
All fibroblasts in culture were negative for epithelial and endothelial markers like E-cadherin and Factor VIII (

| Reprogramming of dermal fibroblasts
As a proof of concept we reprogrammed dermal fibroblasts using a ready-to-use kit and following the well-established protocol supplied with the reagents. 25

| D ISCUSS I ON
Induced pluripotent stem cells are clearly emerging as the most promising cell type in regenerative medicine, as they are autologous cells characterised by pluripotency comparable to that of ESCs.
Nonetheless, several hurdles related to reproducibility and efficiency of reprogramming are yet to be overcome. Fibroblast was the first adult somatic cell to be successfully reprogrammed and thus far it is still the most used cell for reprogramming. Noteworthily, it has been previously demonstrated that fibroblast are not a homogenous population 28 and that cell origin and characteristics affect cell reprogramming. 17  Additionally, while emphasizing fibroblast diversity and the impact of such diversity on cell reprogramming, our study also highlights striking similarity between fibroblasts and MSCs that supports the attracting hypothesis that the most remarkable difference between fibroblasts and MSCs is in the name. [40][41][42][43]57

ACK N OWLED G EM ENTS
This work was supported by a research grant from the Italian Ministry of Education, Universities and Research 20123E8FH4_002.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to disclose.

AUTH O R CO NTR I B UTI O N
AMS and IB contributed equally to this work. FDM and CC are joint senior authors.