Reporter System Controlled by the Involucrin Promoter as a Tool to Follow the Epidermal Differentiation

Myrian Thiago Pruschinski Fernandes University of Sao Paulo (EACH-USP) Jeniffer Farias dos Santos University of Sao Paulo (EACH-USP) Bruna Letícia Freitas University of Sao Paulo (EACH-USP) Gustavo Roncoli Reigado University of Sao Paulo (EACH-USP) Fernanda Antunes University of São Paulo School of Medicine (FM-USP) Nayara Tessarollo University of São Paulo School of Medicine (FM-USP) Felipe Santiago Chambergo University of Sao Paulo (EACH-USP) Bryan Eric Strauss University of São Paulo School of Medicine (FM-USP) Viviane Nunes (  vanunes@ib.usp.br ) University of Sao Paulo (EACH-USP)


) 5 and 7, re
ults in the desquamation process of skin and loss of the nucleus and other cellular organelles.Other pr teins such as cytokeratins (CK) 1, 5, 10 and 14, involucrin, laggrin, loricrin and p63 also participate in the keratinocyte differentiation process 3 .They present speci c expression in the strata of the epidermis, b ing used as markers of epidermal differentiation.Involucrin, a 68 kDa protein, is considered one of the key markers of terminal differentiation of keratinocytes, whose expression begins in the suprabasal layers 4 .

Skin injuries or some pathological conditions may compromise the skin barrier, exposing the organism to external insults.Despite the p operties of healing and wound repair, such as severe burns and diabetes, the skin cannot regenerate 5,6 .Therefore, it is essential to study the molecular mechanisms involved in skin differentiation, both in physiological events and in pathological conditions, in order to meet the emerging demand for developing solutions related to skin in regenerative medicine, tissue bioengineering, gene therapy and in vitro models 7 with high interest for the use of stem cells.Different sources of stem cells have been explored in strategies for skin repair and substitution.Mesenchymal stem cells (MSC) from umbilical cord are adult stem cells that can be obtained by a noninvasive method, which is an advantage when compared to other sourc s of adult stem cells 8, 9 .In addition, they are less immunoreactive 10 and it has been shown that they have immunosuppressive, antiin ammatory, and anti-proliferative properties 11 .However, monitoring the differentiation of these cells in speci c cell types remains a challenge 12 and the existing methods do not allow monitoring of the process over time 13 .

A successful strategy to monitor the differentiation of stem-cell in other lineages is the observation of reporter gene expression directed by promoters that regulate the transcription of speci c genes involved to differentiation 14,15 .Diffe ent methods of transfection have been explored in recent decades, including gene transfer with viruses, which are considered very effective for delivery and integration of genes in MSC.The most used viral vectors in gene transfer are those based on lentiviruses, adenoviruses, baculoviruses and adenoassociated virus 16 .Nunes et al. 9 demonstrated that stem cells obtained from umbilical cord blood transduced with lentivirus achieved an e ciency in transduction of more than 70% of the cells.

Recent studies have investigated the use of speci c gene promoters controlling reporter gene expression to monitor lineage differentiat on 13,14 .Different regulatory promoters of epidermal differentiation proteins have been explored to monitor the differentiation of keratinocytes, such as the CK1 or CK14 promoters 17,18 .However, since involucrin present suprabasal expression, its promoter (pINV) appeared as a more suitable candidate to identify the terminal differentiation of these cells.Another advantage of pINV is that involucrin is expressed in epidermal cells even under conditions in which other markers are not well expressed 19,20,21 .

Considering the potential for the use of MSC in therapeutic applications and in the development of epidermal substitutes, we designed a reporter system containing the uorescent reporter gene EGFP (enhanced green uorescent pro ein) under control of pINV to monitor the differentiation of MSC into keratinocytes in a time-spatial manner.


Results


Obtaining the involucrin promoter

The functional regions of the involucrin promoter, RRP and RRD, with 242 bp and 404 bp, respectively, were ampli ed from genomic HaCaT DNA, as shown in Figure 1A (Supplementary Figure 2).The ampli ed RRD and RRP segments were ligated, at the BamHI restriction site, generating a 659 bp fragment, which corresponds to the pINVf (Figure 1B).The pINVf primers were designed with speci c restriction sites to facilitate cloning (Figure 1C).


Construction and analysis of the recombinant vector

After the construction of the LeGO-GpINV vector (Figure 1D), two strategies were used to con rm the replacement of pSFFV by pINVf.Using the LEGO-F and R primers in PCR experiments, the ampli cation of a 726 bp fragment from the LeGO-G/NeoOpt (control) indicated the presence of pSFFV, and a fragment with 850 bp in LeGO-GpINV (recombinant vec

ed the presence of pINVf.When the
rimers INV-F and R were used, a 659 bp fragment was ampli ed con rming the presence of pINV in the LeGO-GpINV.In the control vector, there was no ampli cation, indicating the absence of pINVf (Figure 1E).According to the digestion strategy, the LEGO-G/NeoOpt vector digested with both NheI and EcoRI enzymes released a fragment with 1960 bp, corresponding to a sequence containing pSFFV, while the recombinant vector was only linearized, ind

ating absence of pSFFV (Figure 1F).

An 850 bp fragm
nt ampli ed from LeGO-GpINV was sequenced.The resulting sequence from the ampli cation with the primer LEGO-R (Supplementary Figure 1) showed 100% identity in the alignment with the predicted sequence.The restriction sites XhoI, BamHI and AscI were identi ed, as well as the essential binding sites (AP1-1, AP1-5, C/EBP and SP1) for involucrin expression, and the cis-acting TATA box region.Replacement of pSFFV with pINVf was also con rmed, and its in-phase insertion and location upstream of EGFP.As the expression of the EGFP reporter protein in LeGO-GpINV is conditioned to the transactivation of pINV during involucrin expression, concentration of the lentiviral production was inferred from LeGO-G/NeoOpt titration by ow cytometry.The titer of the viral preparation was 5.67x10 8 TU/ml (transducing units per ml).


Characterization of MSC

Cultured MSC were characteri ed by ow cytometry using anti-CD105, anti-CD73 and anti-CD90 antibodies, which recognize surface markers present in these cells.As negative controls, MSC were incubated with anti-CD34 and anti-CD31 antibodies (Figure 2), surface antigens present in hematopoietic and endothelial stem cells, respectively.About 99.3% of cells were positive for CD90, 99.6% for CD105 and 98.9% for CD73.MSC incubated with anti-CD34 and CD31 antibodies were not labeled, with only 0.2% and 0.4% of positive events, respectively.HaCaT cells were incubated with CD90 and CD105 (Figure 2B) and a minimum percentage was labeled, 0.5% and 0.3%, respectively, which con rms that the markers are speci c to MSC.


Differentiation of MSC into keratinocytes

For the study and standardization of methods related to the expression of epidermal differentiation markers, pr

iminary assays with non-
ransduced MSC were performed.When cultured in the proliferation medium, MSC possesses a broblast shape (Figure 3A).After 7 days of cultivation in KSFM supplemented with 1.8 mM calcium ions and 5 ng/ml EGF, the cells acquired a polygonal shape and were juxtaposed (Figure 3B, C, D).Furthermore, it is possible to visualize cellular laments, which, although not further investigated, resemble actin laments (indicated by black arrows).


Expression of epidermal differentiation proteins by ow cytometry

The control and differentiated cells were incubated with the speci c antibodies for CK10, CK14 and involucrin and analyzed by ow cytometry (Figure 4).All antibodies were e cient for labeling the proteins of interest.On

C differentiated for 14 days, more than 85
of the cell population expressed CK14, CK10 and involucrin.For CK10, it was possible to identify an increase in the population of cells expressing CK10, as well as in the uorescence intensity, between days 7 and 14.For involucrin, there was an increase in the population of cells expressing involucrin on the seventh day of culture.There was no labeling on the rst day of differentiation or for cultivation with the proliferation medium (negative control).For HaCaT cultivated in DMEM (positive control), 89.4% of the cell population expressed involucrin, 81.2% CK10 and 97.9%

K14.


Detection of the speci c activity of epidermal kallikreins
There was a signi cant and progressive increase in the speci c activity of kallikreins in MSC grown with the differentiation medium compared to those cultured in DMEM (negative control) on the same day (Figure 5A).For hK5, the difference was veri ed for day 1 and for hK6 and 7, the difference was observed on the seventh day of cultivation.Kallikrein activity was signi cantly lower in MSC grown with DMEM and this activity did not change during the time course of cultivation.In differentiated cell cultures, hK5 activity increased 1.8 times on day 14 compared to day 1.For hK6 and 7, the difference occurred between all periods, being 2.0 and 2.6 times higher at day 14 compared to the rst day.In order to ensure that the enzymatic activity observed in the differentiated MSC was related to the presence of epidermal kallikreins, assays were performed with speci c inhibitors for each protease studied.The hK5 activity was completely inhibited by 100 and 500 μM SBTI and 100 μM aprotinin (Figure 5B).This gure also shows that hK7 activity was almost 50% inhibited by 500 μM TPCK.


Expression of involucrin and laggrin by qPCR

The expression of involucrin and laggrin in MSC differentiated into keratinocytes was also studied by RT-qPCR (Figure 6A).When MSC were grown with the differentiation medium, the relative expression of involucrin increased signi cantly on day 7, being 4.2 and 5.1-fold higher on days 7 and 14, respectively, compared to the r

day of cultivation.When cells were grown wit
DMEM, there was no increase in the expression during the analyzed period.In differentiated MSC, laggrin relative expression was 10-fold higher on the seventh day of culture when compared to the rst day, with no signi cant increase between days 7 and 14, nor in cells cultured in DMEM.


Involucrin expression by western blot

The expression of involucrin was higher in MSC cultured in KSFM on days 7 and 14 in comparison to cells cultured in DMEM on the same days (Figures 6B, 6C, Supplementary Figure 3).There was a signi cant increase in the expression of involucrin in MSC on the 14 th day of differentiation compared to the seventh day, which was about 27.2

mes higher than in DMEM in the same pe
iod.As positive control, HaCaT cells cultured in both media were analyzed on the seventh day of the cultivation period, with a 10-fold increase in the involucrin expression in HaCaT cells cultured with KSFM.


Transduction of epidermal cells with lentiviral vectors

The cells were transduced with viral particles produced from LeGO-GpINV and analyzed by inverted uorescence microscopy on days 0, 1, 7 and 14 of differentiation.MSC induced to differentiate into keratinocytes showed a gradual increase in uorescence on days 7 and 14 of the differentiation process.

In MSC growth with DME

(day zero) or non-transduced cultivated with KSFM (contr
l), there was no uorescence (Figure 7).The increase in EGFP expression on days 7 and 14 of culture con rms that the expression of the reporter protein was transactivated by pINVf and that this promoter causes tissue and stratum-speci c expression.

The expression of the reporter protein in the tran duced MSC was also evaluated by ow cytometry.When transduced with the recombinant lentiviral vector, MSC showed an increase in the population of uorescent cells on the seventh day of culture with KSFM.The intensity of uorescence due to EGFP expression was also analyzed, being veri ed the presence of a population of cells that exp ess this protein with high-intensity uorescence (36.7%) on the seventh day of culture, compared to lesser than 5% in cells cultured in DMEM or transduced with the control vector (Figure 7D-II).When transduced with the LeGO-G/NeoOpt vector (MOI of 4), MSC cultured in DMEM (day 0) had 74.62% of cells expressing EGFP, indicating the effectiveness of transduction with the original vector.

HaCaT cells were also transduced to con rm the expression of EGFP controlled by the involucrin promoter in cells that endogenously express the involucrin protein.These cells were cultured in DMEM or KSFM supplemented with 1.8 mM calcium ions and 5 ng/ml EGF, being observed an increase in the population of cells expressing the reporter protein on the seventh day of culture with the differentiation medium.In HaCaT cells cultured in DMEM, there was no difference in the population of cells expressing EGFP (75%) between cells transduced with LeGO-G/NeoOpt or LeGO-GpINV.


Discussion

Different approaches have been explored to monitor and to understand the molecular mechanisms involved in the epidermal differentiation process of MSC into keratinocytes for

th therapeu
ic and biotechnological applications 22,23 .In the present study, we designed a reporter system controlled by pINV as a tool to continuously monitor the epidermal differentiation of MSC.

Before differentiation and transduction experiments, MSC were characterized by immunophenotyping.

According to the International Society for Cellular Therapy 24 , more than 95% of the MSC population must express CD73 (ecto-5′-nucleotidase), CD90 and CD105.Our samples resulted in approx mately 99% of the cells expressing these markers.CD34, a hematopoietic marker, and CD31 (PECAM-1: platelet adhesion molecule), an endothelial marker, were used as negative controls 25 .Speci cally, less than 1% of MSC expressed the negative markers CD31 and CD34.

We have already shown that it is possible to obtain cells with characteristics of keratinocytes by the twodimensional cultivation of MSC in KSFM in the presence of 1.8 mM calcium ions and sup lement containing EGF 5 ng/ml, being veri ed the expression of epidermal markers in a process temporally consistent to one that occurs in human skin 26 .Accordingly, MSC cultivated in these conditions showed typical morphological changes after the seventh day of cultivation, such as changing the broblastoid to polygonal shape like the morphology of keratinocytes in cultures 27 .In addition, it was possible to observe structures that resemble actin laments, characteristic of suprabasal keratinocytes 28 .

The current method to induce epidermal transdifferentiation of MSC resulted in an expressive population of cells expressing CK14, CK10 and involucrin after 14 days of culture (higher than 85%).
he high expression of involucrin and laggrin after 14 days of differentiation also indicate that most cells were terminally differentiated.

Among the several factors that in uence the proliferation and differentiation of keratinocytes 29 , the concentration of calcium ions has a great in uence on the regulation of epidermal differen iation and the expression of different epidermal markers, such as involucrin and hK 30 .One of the pathways for regulating the expression of epidermal differentiation markers is the PKC pathway, which is strongly activated by calcium.The extracellular calcium gradient increase leads to the enhancement of transcription factors ligation to its respective binding sites present in pINV 31 .However, there are controversies in the action of EGF to induce epidermal differentiation 29,32 .

In addition to demonstrating the ability of MSC to transdifferentiate into keratinocytes, it was essential to show the expression of involucrin in cells induced to differentiate simultaneously with the expression of the reporter protein.In MSC induced to differentiation, increased expression of involucrin was identi ed on days 7 and 14 of culture and laggrin on day 7.In contrast, in cells cultivated in the proliferation medium, there was no expression of these proteins.By ow cytometry, approximately 100% of MSC cultured with differentiation medium, for 7 days, were labeled with anti-involucrin antibody.This expression persisted, being identi ed on the 14th day of cultivation, which indicates that most of the cell population reached terminal differentiation.These data agree with previous studies 5,6,26 and corroborate the effectiveness of the proposed protocol to differentiate MSC in keratinocytes.

Several authors have already differentiated MSC in epidermal cells under different culture conditions and evaluated the involucrin expression, which indicates the terminal differentiation.However, our met od showed very effective compared to other studies 6, 33, 34 , obtaining a high population of cells expressing involucrin after 14 days and higher relative expression of involucrin mRNA.In the present study, the expression of involucrin and laggrin mRNA was 5 and 10 times higher, respectively, after 14 days of culture with differentiation medium when compared to cells cultured in DMEM.When evaluated by western blot, the expression of involucrin in differentiated MSC was 27 times higher than in cells cultured in DMEM in the same period, and about 3 times higher than in HaCaT cells cultured in this medium.

In addition to the assessment of epidermal marker expression, MSC differentiation into keratinocytes was evaluated by the detection of the speci c enzyme activity of tissue kallikreins on the hydrolysis of s eci c FRET substrates.Tissue kallikreins are considered attractive targets for the development of new therapies involving the airways, structures of the cardiovascular system, teeth, brain, skin and neoplastic diseases 35 .Also, recently, they appear as innovative targets for the treatment of some skin diseases, such as Netherton's syndrome 36 , with different approaches being proposed to detect and inhibit their activity.The tissue kallikrein activity measured in cell extracts was compatible with those of Santos et al. 26 , with a progressive increase in the activity on days 7 and 14 of cultivation of MSC with the differentiation medium.Additional assays were performed using speci c inhibitors of trypsin and chymotrypsin-like activity, and the activity of hK5 e 7 was inhibited by the tested inhibitors, according to Goettiga et al. 37 .

In this study, we established a monitoring system that allows us to trace the differentiation of MSCs into keratinocytes by EGFP expression under the control of pINV.Therefore, that tool will allow monitoring th differentiation in a temporal, accurate and easy way, evaluating the effect of different chemical and/or physical stimulation in the epidermis.The pINVf was successfully ampli ed and cloned, maintaining the AP1-1, AP1-5, C/EBP and SP1 binding sites, as con rmed by sequencing, which are essential for the involucrin expression 31,38,39 .Comparing to other epidermal promoters, such as the pCK14 40 , pINVf has been proved to be a good candidate for viral vector production, since promoters larger than 1.5 Kb fail to deliver tissue-speci c genes and can result in reduced viral titer and is able to promote high expression of the reporter protein 17 .

Different studies have shown that lentiviral is e cient as a gene delivery method in MSC transduction.

Nunes et al. 9 achieved a transduction e ciency greater than 70% in MSC from umbilical cord blood with lentivi us encoding EGFP.Zhang et al. 41 demonstrated that MSC from bone marrow transduced with lentivirus show d a proportion of transduced cells expressing the EGFP and DsRed reporter proteins higher than 75% using MOI below 1.0 and persistent expression for more than 5 months.It has also been

shown that viral transduction of MSC can reach up to 90% of cells, without affecting the quality of progeny and the differentiation capacity of these cells 42,43 .

In the LeGO-GpINV construction, the antibiotic resistance (neomycin) and the reporter gene are downstream from pINVf, which restrict its transactivation to cells that express involucrin and not in nondifferentiated ells.Therefore, the titration and the MOI assays was performed with the LeGO-G/NeoOpt preparation and the viral titration and MOI was extrapolated to the recombinant vector preparations.Although, our results demonstrated that HaCaT cells transduced with LeGO-GpINV had similar transduction e ciency to the HaCaT cells transduced with LeGO-G/NeoOpt vector.

Both cell types were transduced in suspension with MOI 4 with concentrated viral preparation and the e ciency of approximately 75% of cells expressing EGFP was achieved in MSC transduced with the LeGO-G/NeoOpt vector and in HaCaT cells transduced with LeGO-G/NeoOpt or LeGO-GpINV.In addition, more than half of the differentiated MSC population expressed EGFP on the seventh day of culture, compared with 75% of positive cells in the transduction of MSC with the LeGO-G/NeoOpt vector.

Accordingly, it was shown an increase in the green uorescence intensity corresponding to the expression of EGFP on days 7 and 14 of differentiation of MSC transduced with LeGO-GpINV.In parallel, it was veri ed, by ow cytometry, an increase in the population of cells expressing involucrin on day 7.In nondifferentiated MSC transduced with LeGO-GpINV, t e expression of the reporter protein was not detected, con rming the speci city of the expression of EGPF.These results demonstrate that pINVf was able to promote stratum-speci c expression of EGFP, simultaneously with the expression of involucrin in these cells.The results obtained corroborate with previous studies 6, 44,45 , where it was demonstrated that the involucrin promoter is active in the cells that express this protein.

Collectively, the results show that KSFM supplemented with calcium and EGF ions was e cient to induce the transdifferentiation of MSC into keratinocytes and that these cells showed expression of differentiation epidermal markers over 14 days of cultivation.It was also veri ed that the EGFP expression coincides with the expression of involucrin in these cells, demonstrating that the tool is functional and e cient to monitor the terminal differentiation of keratinocytes.In

reporter system proved to be very p
omising in a differentiation model of MSC in keratinocytes in vitro.This tool can be extended to 3D skin models, opening perspectives to be used as a simple and low-cost option to monitor cells in vivo.In addition, it can enable a better understanding of skin biology, both in physio and pathological conditions or for MSC use in tissue bioengineering and in regenerative medicine.


Methods


Construction of recombinant vectors

To obtain the involucrin promoter, the RRD and RRP functional regions of pINV, as previous described 17,38 , were ampli ed using oligonucleotides synthesized with speci c restriction sites, as shown in Table 1, from the genomic DNA of HaCaT cells, extracted with QIAamp DNA Stool Mini Kit (QIAgen, Hilden, Germany).The minimal functional involucrin promoter (pINVf), a 659-bp segment, was obtained through ligation of RRD and RRP in BamHI restriction site, followed by ampli cation.The ampli cations were performed by polymerase chain reaction (PCR) using Taq Platinum kit (Invitrogen, Waltham, MA), following the program: 95ºC for 5 min; 35 cycles of 95ºC for 30 sec, 55ºC for 30 sec, 72ºC for 1 min; 72ºC for 10 min.The pINVf segment was inserted into the lentiviral vector LeGO-G/NeoOpt (46 between the XhoI and AscI restriction sites, upstream the EGFP, replacing the spleen focus-forming virus (pSFFV) virus promoter.OneShot® Stbl3TM (Invitrogen, Waltham, MA) competen

E. coli were used for
vector DNA ampli cation.

To con rm the correct construction and absence of pSFFV in the vector, two PCR ampli cation reactions were performed using two sets of primers (Table 1): 1) LEGO-F and LEGO-R, which are located upstream and downstream of the pSFFV sequence, respectively; and 2) INV-F and INV-R.The presence of the pINVf sequence and the absence of pSFFV was also con rmed by digestion of the vectors with the enzymes EcoRI and NheI, considering that the NheI site is present only in the vector LeGO-G/NeoOpt, upstream of the pSFFV.The DNA sequencing covered the anking regions of the pINVf insertion site in the lentiviral vector.A fragment was ampli ed using the primer set LEGO-F and LEGO-R (Table 1).Subsequently it was sequenced using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scienti c, Waltham, MA) and DNA sequencing reaction was carried out in an ABI 3730 DNA Analyzer (Applied Biosystems, Waltham, MA) by capillary electrophoresis using the Sanger sequencing method 47

Lentiviral packaging

Lentiviral particles we
e packed using a 2nd generation packaging system.Plasmids LeGO-G/NeoOpt, pCMV-VSVg envelope and psPAX2 were mixed, and transfection of HEK 293T cells (2 x 10 6 ) was performed using the calcium phosphate precipitation method, ada

ed from Naviaux et al. 48.Lentiviral supernat
nts were collected 24 and 48 h after transfection, ltered through a 0.45 μm pore size syringe lter and then concentrated by ultracentrifugation at 110,000 x g for 90 min at 4ºC in the SW 32 Ti Rotor (Beckman Coulter, Brea, CA).The pellets were resuspended in 100 μl of Hank's Balanced Salt Solution (HBSS -Life Technologies, CA) and incubated at 4ºC for 24 h.Solutions were stored at -80ºC.The lentivirus titration was performed in HT1080 cells (5x10 4 ), a human brosarcoma cell line.The cells were incubated with the viruses for 6 to 8 h and subsequently the expression of the reporter protein was analyzed by ow cytometry in a FACSCALIBUR cytometer (Becton Dickinson, Franklin Lakes, NJ) at λ = 530 nm.The r

ults were obtained in TU/ml (transducing units per ml
and virus titers > 10 6 per ml (for non-concentrated supernatants) were obtained, as expected 49 .


Cell cultures

MSC from the umbilical cord were obtained as described by Santos et al 26  fetal bovine serum (FBS) (Gibco, Grand Island, NY), 10,000 U/ml penicillin, 100 mg/ml streptomycin (Invitrogen, Waltham, MA), in a humidi ed atmosphere and 5% CO 2 at 37°C.MSC were used in the passages from 2 to 10 as indicated in the different experiments.


Characterization of MSC by ow cytometry

MSC immunophenotyping was performed according to the expression of cell surface markers detected by ow cytometry.Five thousand cells per sample were evaluated on the Guava EasyCyte™ system (Merck Millipore, Burlington, MA) using a blue argon laser (488 nm) and readings were performed at λ=525/30 nm, λ=583/26 nm and λ=695/50 nm.The cells wer

elation to the total
umber of events.

In vitro differentiation of MSC into keratinocyte-like cells MSC were distributed in 6-well plates and cultured in proliferation medium until reaching 80% con uence.Induction of MSC to differentiate into keratinocyte lineage was performed by culturing in Keratinocyte Serum Free Medium (KSFM) (Gibco, Grand Island, NY) supplemented with 1.8 mM calcium ions and the speci c supplement De ned Keratinocyte-SFM Growth Supplement containing 5 ng/ml EGF.The cells were analyzed before changing the culture medium to KSFM (day zero) and after 1, 7 and 14 days of culture with this medium.The morphological changes were accompanied by optical microscopy in a Nikon Eclipse TS100 inverted microscope (Nikon Instruments Inc., USA).


Expression of epidermal markers

The expression of epidermal differentiation markers was evaluated by ow cytometer, qPCR and western blot in MSC induced to differentiation after different incubation times (0,