High-efficiency production of bioactive recombinant human fibroblast growth factor 18 in Escherichia coli and its effects on hair follicle growth

Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-β-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.     

Expression and purification of an FGF9 fusion protein in E. coli,and the effects of the FGF9 subfamily on human hepatocellular carcinoma cell proliferation and migration

Fibroblast growth factor (FGF) 9 has oncogenic activity and plays an important role in the development of ovarian, lung, prostate, and gastric cancers. In the present study, with the aim of reducing the cost of utilizing growth factors in cancer research, a simple and efficient method for the preparation of recombinant human (rh)FGF9 in Escherichia coli was established. The rhFGF9 fusion protein (6 × His-TEV-rhFGF9) and the native protein released by tobacco etch virus (TEV) protease were obtained using a Ni-NTA system, with > 95% purity. Both purified forms of rhFGF9, with and without fusion tags, significantly stimulated the proliferation of NIH3T3 cells. The FGF9 subfamily, including FGF9, FGF16, and FGF20, in addition to rhFGF16, rhFGF9, and rhFGF20, were shown to stimulate the proliferation and migration of HuH7 human hepatocellular carcinoma (HCC) cells. Mechanistic studies revealed that the stimulation of HuH7 cell proliferation and migration with rhFGF9 and rhFGF20 were associated with the activation of the extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) pathways and matrix metalloproteinase-26 (MMP26). Inhibition of the ERK and NF-κB pathways blocked cell migration, and NF-κB was demonstrated to be regulated by ERK. Therefore, the present study demonstrates a simple method for the preparation of biologically active rhFGF9 protein. Furthermore, the results indicate that exogenous rhFGF9- and rhFGF20-activated ERK/NF-κB signal transduction pathways play important roles in the regulation of HCC cell proliferation and migration, and this discovery helps to find the potential for new solutions of the treatment of liver cancer.

     

High-yield of biologically active recombinant human fibroblast growth factor-16 in E. coli and its mechanism of proliferation in NCL-H460 cells

Fibroblast growth factor-16 (FGF16) is a member of FGF9 subfamily, which plays key role in promoting mitosis and cell survival, and also involved in embryonic development, cell growth, tissue repair, morphogenesis, tumor growth, and invasion. However, the successful high-yield purification of recombinant human fibroblast growth factor-16 (rhFGF16) protein has not been reported. In addition, lung cancer is a major cause of cancer-related deaths, which threats people’s lives and its incidence has continued to rise. Learning pathways or proteins, which involved in lung tumor progression will contribute to the development of early diagnosis and targeted therapy. FGF16 promoted proliferation and invasion behavior of SKOV-3 ovarian cancer cells, whose function may be similar in lung cancer. The hFGF16 was cloned into pET-3d and expressed in Escherichia coli BL21 (DE3) pLysS. Finally, obtained two forms of FGF16 that exhibited remarkable biological activity and the purity is over 95%, meanwhile, the yield of soluble 130?mg/100?g and insoluble 240?mg/100?g. Experiments demonstrated FGF16 could promote proliferation of NCL-H460 cells by activating Akt, Erk1/2, and p38 MAPK signaling, whereas JNK had no significant effect. In total, this optimized expression strategy enables significant quantity and activity of rhFGF16, thereby meeting its further pharmacological and clinical usages.     

A better anti-diabetic recombinant human fibroblast growth factor 21 (rhFGF21) modified with polyethylene glycol

As one of fibroblast growth factor (FGF) family members, FGF21 has been extensively investigated for its potential as a drug candidate to combat metabolic diseases. In the present study, recombinant human FGF21 (rhFGF21) was modified with polyethylene glycol (PEGylation) in order to increase its in vivo biostabilities and therapeutic potency. At N-terminal residue rhFGF21 was site-selectively PEGylated with mPEG20 kDa-butyraldehyde. The PEGylated rhFGF21 was purified to near homogeneity by Q Sepharose anion-exchange chromatography. The general structural and biochemical features as well as anti-diabetic effects of PEGylated rhFGF21 in a type 2 diabetic rat model were evaluated. By N-terminal sequencing and MALDI-TOF mass spectrometry, we confirmed that PEG molecule was conjugated only to the N-terminus of rhFGF21. The mono-PEGylated rhFGF21 retained the secondary structure, consistent with the native rhFGF21, but its biostabilities, including the resistance to physiological temperature and trypsinization, were significantly enhanced. The in vivo immunogenicity of PEGylated rhFGF21 was significantly decreased, and in vivo half-life time was significantly elongated. Compared to the native form, the PEGylated rhFGF21 had a similar capacity of stimulating glucose uptake in 3T3-L1 cells in vitro, but afforded a significantly long effect on reducing blood glucose and triglyceride levels in the type 2 diabetic animals. These results suggest that the PEGylated rhFGF21 is a better and more effective anti-diabetic drug candidate than the native rhFGF21 currently available. Therefore, the PEGylated rhFGF21 may be potentially applied in clinics to improve the metabolic syndrome for type 2 diabetic patients.     

Stimulation of mouse vibrissal follicle growth by recombinant human fibroblast growth factor 20

Objectives

To explore potential effects of recombinant human fibroblast growth factor 20 (rhFGF20) in the growth of cultured mouse vibrissal follicles.

Results

The growth of cultured mouse vibrissal follicles was significantly induced by rhFGF20 in a dose dependent pattern in the in vitro vibrissal follicle organ culture model. However, too high concentration of rhFGF20 could inhibit the growth of vibrissal follicles. We further demonstrated that rhFGF20 stimulated the proliferation of hair matrix cells and activated Wnt/β-catenin signaling pathway.

Conclusions

The rhFGF20 might be a potential therapeutic agent to treat hair loss disorders.

     

Isolation,purification and characterization of a pH tolerant and temperature stable proteinaceous protease inhibitor from marine <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis>

Objectives

An extracellular protease inhibitor (BTPI-301) of trypsin was purified and characterized from an isolate of Pseudomonas mendocina.

Results

BTPI-301was purified to homogeneity by (NH₄)₂SO₄, precipitation, DEAE Sepharose and CNBr-activated Sepharose chromatography. Homogeneity was proved by native PAGE and SDS-PAGE. The intact molecular mass was 11567 Da by MALDI-TOF analysis. BTPI-301was a competitive inhibitor with a Ki of 3.5 × 10^?10 M. It was stable and active at pH 4–12 and also at 4–90 °C for 1 h. Peptide mass fingerprinting by MALDI revealed that the BTPI-301 is a new inhibitor not reported so far with protease inhibitory activity. The pI of the inhibitor was 3.8. The stoichiometry of trypsin-BTPI-301 interaction is 1:1. The inhibitor was specific towards trypsin.

Conclusion

A pH tolerant and thermostable protease inhibitor BTPI-301 active against trypsin was purified and characterized from P. mendocina that could be developed and used as biopreservative as well as biocontrol agent.

     

Injection of FGF6 accelerates regeneration of the soleus muscle in adult mice

FGF6, a member of the fibroblast growth factor (FGF) family, accumulated almost exclusively in the myogenic lineage, supporting the finding that FGF6 could specifically regulate myogenesis. Using FGF6 (-/-) mutant mice, important functions in muscle regeneration have been proposed for FGF6 but remain largely controversial. Here, we examined the effect of a single injection of recombinant FGF6 (rhFGF6) on the regeneration of mouse soleus subjected to cardiotoxin injection, specifically looking for molecular and morphological phenotypes. The injection of rhFGF6 has two effects. First, there is an up-regulation of cyclin D1 mRNA, accounting for the regulating role of a high FGF6 concentration on proliferation, and second, differentiation markers such as CdkIs and MHC I and Tn I increase and cellular differentiation is accelerated. We also show a down-regulation of endogenous FGF6, acceleration of FGFR1 receptor expression and deceleration of the FGFR4 receptor expression, possibly accounting for biphasic effects of exogenous FGF6 on muscle regeneration.     

Cytotoxicity and Glycan-Binding Profile of a <Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Binding Lectin from the Eggs of a Japanese Sea Hare (<Emphasis Type="Italic">Aplysia kurodai</Emphasis>)

A divalent cation-independent 16 kDa d-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized d-galactose and d-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl d-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k _ass and k _diss values are 2.4 × 10³ M^?1 s^?1 and 3.8 × 10^?3 s^?1, respectively. AKL-2 appeared cytotoxicity against both Burkitt’s lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.     

Relationship between TRAF6 and deterioration of HCC: an immunohistochemical and in vitro study

Objective

To explore the relationship between tumor necrosis factor receptor-associated factor 6 (TRAF6) and the clinicopathological features in HCC as well as its biological function.

Methods

Totally, 412 liver tissues were collected, including 171 hepatocellular carcinoma (HCC) and their corresponding non-tumor tissues, 37 cirrhosis and 33 normal liver tissues. The expression of TRAF6 was assessed by immunohistochemistry. Then, analysis of the correlations between TRAF6 expression and clinicopathological parameters in HCC was conducted. Furtherer, in vitro experiments on HepG2 and Hep3B cells were performed to validate the biological function of TRAF6 on HCC cells. TRAF6 siRNA was transfected into HepG2 and Hep3B cell lines and TRAF6 expression was evaluated with RT-qPCR and western blot. The assays of cell viability, proliferation, apoptosis and caspase-3/7 activity were carried out to investigate the effects of TRAF6 on HCC cells with RNA interference. Cell viability was assessed with Cell Titer-Blue kit. Cell proliferation was tested with MTS kit. Cell apoptosis was checked through morphologic detection with fluorescence microscope, as well as caspase-3/7 activity was measured with fluorogenic substrate detection.

Results

The positive expression rate of TRAF6 protein was 49.7 % in HCC, significantly higher than that of normal liver (12.1 %), cirrhosis (21.6 %) and adjacent non-cancerous tissues (36.3 %, all P < 0.05). Upregulated TRAF6 was detected in groups with metastasis (Z = ?2.058, P = 0.04) and with low micro-vessel density (MVD) expression (Z = ?2.813, P = 0.005). Spearman correlation analysis further showed that the expression of TRAF6 was positively correlated with distant metastasis (r = 0.158, P = 0.039) and negatively associated with MVD (r = ?0.249, P = 0.004). Besides, knock-down of TRAF6 mRNA in HCC cell lines HepG2 and Hep3B both resulted in cell viability and proliferation inhibition, also cell apoptosis induction and caspase-3/7 activity activation.

Conclusions

TRAF6 may contribute to metastasis and deterioration of the HCC via influencing cell growth and apoptosis. Thus, TRAF6 might become a predictive and therapeutic biomarker for HCC.

     

Purification,characterization and fine sugar specificity of a N-Acetylgalactosamine specific lectin from <Emphasis Type="Italic">Adenia hondala</Emphasis>

Plant lectins are gaining interest because of their interesting biological properties. Several Adenia species, that are being used in traditional medicine to treat many health ailments have shown presence of lectins or carbohydrate binding proteins. Here, we report the purification, characterization and biological significance of N-Acetyl galactosamine specific lectin from Adenia hondala (AHL) from Passifloraceae family. AHL was purified in a single step by affinity chromatography on asialofetuin Sepharose 4B column, characterized and its fine sugar specificity determined by glycan array analysis. AHL is human blood group non specific and also agglutinates rabbit erythrocytes. AHL is a glycoprotein with 12.5% of the carbohydrate, SDS-PAGE, MALDI-TOF-MS and ESI-MS analysis showed that AHL is a monomer of 31.6 kDa. AHL is devoid of DNase activity unlike other Ribosome inactivating proteins (RIPs). Glycan array analysis of AHL revealed its highest affinity for terminal lactosamine or polylactosamine of N- glycans, known to be over expressed in hepatocellular carcinoma and colon cancer. AHL showed strong binding to human hepatocellular carcinoma HepG2 cells with MFI of 59.1 expressing these glycans which was effectively blocked by 93.1% by asialofetuin. AHL showed dose and time dependent growth inhibitory effects on HepG2 cells with IC₅₀ of 4.8 μg/ml. AHL can be explored for its clinical potential.     

Fibroblast growth factor 2‐antagonist activity of a long‐pentraxin 3‐derived anti‐angiogenic pentapeptide

Fibroblast growth factor‐2 (FGF2) plays a major role in angiogenesis. The pattern recognition receptor long‐pentraxin 3 (PTX3) inhibits the angiogenic activity of FGF2. To identify novel FGF2‐antagonistic peptide(s), four acetylated (Ac) synthetic peptides overlapping the FGF2‐binding region PTX3‐(97–110) were assessed for their FGF2‐binding capacity. Among them, the shortest pentapeptide Ac‐ARPCA‐NH₂ (PTX3‐[100–104]) inhibits the interaction of FGF2 with PTX3 immobilized to a BIAcore sensorchip and suppresses FGF2‐dependent proliferation in endothelial cells, without affecting the activity of unrelated mitogens. Also, Ac‐ARPCA‐NH₂ inhibits angiogenesis triggered by FGF2 or by tumorigenic FGF2‐overexpressing murine endothelial cells in chick and zebrafish embryos, respectively. Accordingly, the peptide hampers the binding of FGF2 to Chinese Hamster ovary cells overexpressing the tyrosine‐kinase FGF receptor‐1 (FGFR1) and to recombinant FGFR1 immobilized to a BIAcore sensorchip without affecting heparin interaction. In all the assays the mutated Ac‐ARPS A‐NH₂ peptide was ineffective. In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF‐binding domain of the Ig‐like loop D2 of FGFR1, amino acid substitutions in Ac‐ARPCA‐NH₂ and saturation transfer difference‐nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding. These results will provide the basis for the design of novel PTX3‐derived anti‐angiogenic FGF2 antagonists.     

Purification and Characterization of a Major Cell Surface Glycoprotein in Zajdela Ascites Hepatoma Cells Which Displays a Potent Concanavalin A Receptor Activity

A major cell surface sialoglycoprotein with Concanavalin A receptor activity has been isolated from rat Zajdela ascites hepatoma cells. The sialic acid residues of the plasma membrane glycoproteins were specifically labeled by oxidation with NaIO₄ followed by reduction with NaB³H₄. Surface-labeled glycoproteins were released by short incubations with TPCK-trypsin at 37°C and then separated by gel filtration on Sepharose 6B column. The predominantly labeled fraction, GP II₂, was then purified by chromatography on DEAE-cellulose equilibrated with 0.05 M phosphate buffer, pH 7.5, and eluted with increasing molarities of NaCl. It was shown to be homogeneous by protein and carbohydrate staining on SDS-polyacrylamide gels, isoelectric focusing, rechromatography on DEAE-cellulose and immunoelectrophoresis. It has an apparent molecular weight of 110,000 daltons. The location of GP II₂on the cell surface was confirmed by the fact that it could be labeled metabolically with, D-(³H) glucosamine and externally through the nonpenetrating periodate-NaB³H₄ system. GP II₂could not be removed from the cell surface by high salt concentrations, chelator, or chaotropic agents but was released from the membrane by detergents. This suggests that GP II₂could be an integral protein. Analysis of the carbohydrate composition of GP II₂ revealed galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid as major constituents and mannose as a minor one. This suggests that it contains carbohydrate chains both O- and N-linked to the polypeptide chain, most of them being O-linked. Finally, GP II₂has a potent Concanavalin A receptor activity. It inhibits the interaction between Concanavalin A and hepatoma cells and suppresses its effects on hepatoma cell proliferation.     

The plaque-forming cell response of human blood lymphocytes. I. PFC response of lymphocytes to formalin-treated staphylocci.

The plaque-forming cell and proliferative responses of human peripheral blood lymphocytes induced by formalin-treated Staphylococcus aureus of the Cowan strain were studied in vitro. Human blood mononuclear cells were incubated for 6 days with staphylococci in culture medium RPMI 1640 supplemented with 10% human AB serum. The number of anti-sheep erythrocyte plaque-forming cells was determined by the Jerne technique. Lymphocyte proliferation was measured by [³H]thymidine incorporation. Individual lymphocyte donors could be classified as high or low responders to staphylococci. Lymphocyte proliferation appeared necessary for the generation of plaque-forming cells. The plaque-forming cell response was greatly influenced by the source of the human AB serum used in the culture medium. The addition of hydrocortisone to the culture medium augmented the plaque-forming cell response. Human B lymphocytes prepared by passage through a column containing Sepharose 4B conjugated to anti-human F(ab)₂ generated plaque-forming cells when incubated with staphylococci. However, the addition of T lymphocytes to these B-lymphocyte preparations augmented the plaque-forming cell response to staphylococci.     

Hydrocarbon-coated sepharoses. Use in the purification of glycogen phosphorylase

A homologous series of hydrocarbon-coated Sepharoses varying in the length of their alkyl side chains (Seph-NH(CH₂)_nH) was synthesized. These modified Sepharoses provide a versatile tool for the purification of proteins, since, by choosing the suitable member of the series, a desired protein can be extracted from a protein mixture. This is illustrated in the case of glycogen phosphorylase, which is not retained at all by methyl Sepharose (n=1), is retarded by propyl Sepharose (n=3), is adsorbed on butyl Sepharose (n=4) and can be eluted from the column by deforming buffers, and is so tightly adsorbed on hexyl Sepharose (n=6) that it could be eluted from the column only in the denatured form, by washing with 0.2 N CH₃COOH. On a preparative scale, a hundred-fold purification of phosphorylase could be achieved in one step, by passage of a crude muscle extract on a small butyl Sepharose column.     

A Calcium Ion-Dependent Dimeric Bean Lectin with Antiproliferative Activity Toward Human Breast Cancer MCF-7 Cells

In this study, a 60.8-kDa dimeric lectin was isolated from the Phaseolus vulgaris cv. jade bean and characterized. The lectin was bound on Blue Sepharose 6 and Q Sepharose and was finally purified by size exclusion chromatography on Superdex 200. Its hemagglutinating activity toward rabbit erythrocytes was dependent on divalent cations, especially calcium ions. Various carbohydrates tested were devoid of any effect on the hemagglutinating activity. The lectin was stable at pH between 4.5 and 9.4 and temperatures between 30 and 70 °C. It did not exert antifungal activity toward Valsa mali, Setosphaeria turcica, Mycosphaerella arachidicola, Fusarium oxysporum and Bipolaris maydis. The IC₅₀ of the antiproliferative activity of the lectin toward MCF-7 human breast cancer cells was 174 μM. It did not inhibit proliferation of WRL-68 human normal embryonic hepatocytes. The lectin was dependent on calcium ions for hemagglutinating activity and possessed a blocked N-terminus. These two characteristics make the lectin unique among Phaseolus lectins.     

Cationic Antimicrobial Peptides Derived from <Emphasis Type="Italic">Crocodylus siamensis</Emphasis> Leukocyte Extract,Revealing Anticancer Activity and Apoptotic Induction on Human Cervical Cancer Cells

Known antimicrobial peptides KT2 and RT2 as well as the novel RP9 derived from the leukocyte extract of the freshwater crocodile (Crocodylus siamensis) were used to evaluate the ability in killing human cervical cancer cells. RP9 in the extract was purified by a combination of anion exchange column and reversed-phase HPLC, and its sequence was analyzed by mass spectrometry. The novel peptide could inhibit Gram-negative Vibrio cholerae (clinical isolation) and Gram-positive Bacillus pumilus TISTR 905, and its MIC values were 61.2 µM. From scanning electron microscopy, the peptide was seen to affect bacterial surfaces directly. KT2 and RT2, which are designed antimicrobial peptides using the C. siamensis Leucrocin I template, as well as RP9 were chemically synthesized for investigation of anticancer activity. By Sulforhodamine B colorimetric assay, these antimicrobial peptides could inhibit both HeLa and CaSki cancer cell lines. The IC₅₀ values of KT2 and RT2 for HeLa and CaSki cells showed 28.7–53.4 and 17.3–30.8 µM, while those of RP9 were 126.2 and 168.3 µM, respectively. Additionally, the best candidate peptides KT2 and RT2 were used to determine the apoptotic induction on cancer cells by human apoptosis array assay. As a result, KT2 and RT2 were observed to induce apoptotic cell death in HeLa cells. Therefore, these results indicate that KT2 and RT2 with antimicrobial activity have a highly potent ability to kill human cervical cancer cells.     

Expression of scleraxis and tenascin C in equine adipose and umbilical cord blood derived stem cells is dependent upon substrata and FGF supplementation

Recovery from tendon injury is based on long periods of rest, which results in sub-optimal repair, often replacing tendon with fibrocartilage scar tissue. Recently, the use of stem cells in equine tendon repair has been attempted with variable success. The objective of this work was to determine the expression of scleraxis (scx) and tenascin C (TnC), two markers of tenocytes, in adipose (AdMSC) and umbilical cord blood (UCB) stem cells during culture on various substrata and in response to fibroblast growth factor (FGF) treatment. Equine UCB and AdMSC were cultured on gelatin-coated plasticware, 30 % matrigel or collagen-coated Cytodex beads and treated with 10 ng/ml FGF2, FGF4 or FGF5 prior to measurement of proliferation, kinase activity and tenocyte gene expression. Supplementation with FGF2 or FGF5 activated the ERK1/2 signaling pathway in AdMSC and UCB; no effect of FGF4 was observed in UCB. FGF2 increased proliferation in AdMSC but not UCB. Conversely, FGF5 stimulated proliferation of UCB. Culture in matrigel increased scx expression in both cell populations and increased TnC in AdMSC. In AdMSC grown in matrigel, supplementation with FGF2 or FGF5 increased TnC expression. Thus, culture conditions (substrata and FGF supplementation) impact markers of tenocytes in AdMSC and UCB stem cells, indicating that careful consideration should be given to culture conditions prior to use of UCB or AdMSC as therapeutic aids. Optimal culture conditions may promote early differentiation of these cells, improving their ability to aid tendon regeneration and facilitating more efficient recovery from tendon injury.