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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/066—Tenocytes; Tendons, Ligaments
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/19—Growth and differentiation factors [GDF]

Definitions

• the present invention relates to a culture medium for promoting human bone marrow mesenchymal stem cells differentiation into tenogenic lineage.
• the present invention further relates to a method to differentiate human bone marrow mesenchymal stem cells into tenogenic lineage.
• MSCs Mesenchymal stem cells
• hMSCs tissue engineering approaches are presently being developed to induce tenogenic differentiation in human MSCs (hMSCs) or to produce controlled differentiation of hMSCs into the desired tenogenic lineage prior to transplantation.
• growth differentiation factor-5 which is a member of human bone morphogenetic protein (BMP) family, has been identified as a key biological molecule which can accelerate tendon healing (Aspenberg et ah, 1999). Although the use of GDF-5 have shown to alter tendon formation rate and function in vivo (Aspenberg et al., 1999), little has been described about the effects of GDF-5 on hMSCs proliferation and differentiation in vitro.
• the present invention addresses the use of GDF-5 as an induction factor and other optimal culture condition for hMSC proliferation and tenogenic differentiation in vitro. This may be important when considering that the results may provide a reference data for MSCs culture conditions in laboratory, which can lead to a wider implication for clinical applications.
• An object of the present invention is to provide a culture medium for promoting human bone marrow mesenchymal stem cells differentiation into tenogenic lineage.
• the culture medium not only initiated tenogenic transformation in MSCs but also maintains the production of tendon matrix substances which is inherently important for tendon repair.
• a further object of the present invention is to provide a method to differentiate human bone marrow mesenchymal stem cells into tenogenic lineage.
• FIG. 1 Alamar Blue (AB) cell proliferation assay for hMSCs cultured in different concentration of GDF-5 (ng/ml). Cell proliferation in hMSCs showed no significant differences at varying concentration of GDF-5.
• AB Alamar Blue
• Figure 2 Total collagen content analysis for dose response of hMSCs treated with different concentration of GDF-5.
• FIG. 3 Gene expression analysis of candidate tenogenic markers: type-I collagen (Col-I) scleraxis (Sex), type-Ill collagen (Col- ⁇ ), decorin (Dec, and nucleostemin (Nst) in hMSCs treated with GDF-5 (0, 50 and 100 ng/ml).
• Col-I, Sex and Col-III showed significant up-regulation (*p ⁇ 0.05 and **p ⁇ 0.01), at 100 ng/ml of GDF-5.
• the mononuclear layer (second top layer) was then collected and washed twice with Dulbecco's Modified Eagle's Medium (DMEM) low glucose (DMEM-LG) (Invitrogen-Gibco, USA) supplemented with antibiotic/antimycotic 1% (v/v) (Invitrogen-Gibco, USA).
• DMEM Dulbecco's Modified Eagle's Medium
• DMEM-LG Dulbecco's Modified Eagle's Medium
• DMEM-LG Dulbecco's Modified Eagle's Medium
• DMEM-LG low glucose
• antibiotic/antimycotic 1% Invitrogen-Gibco, USA
• the isolated mononuclear cells were cultured in growth medium (DMEM-LG supplemented with 10% FBS, antibiotic/antimycotic 1% (v/v) and 2 mM L-glutamin; all from Invitrogen-Gibco (USA) and transferred into T75 tissue culture flask
• fibroblastic like cell colonies formed in these cultures demonstrated a low degree of heterogeneity.
• the numbers of fibroblastic appearance like cells became increasingly noticeable over time while in contrast, the heterogeneous cells (such as polygonal cells) became lesser.
• adherent fibroblastic hMSCs isolated from human bone marrow grew in size and numbers while the non-adherent cells were quickly removed when media changes were performed which happened every three-days.
• the final homogeneous cell population obtained at end of passage 2 (P2) cell culture was used for downstream differentiation assay.
• Isolated cells appear to conform to the characteristics expected of MSCs. Cells appears to have (1) spindle shaped plastic adherent features, (2) demonstrated positive markers for CD45, CD105 and CD166 while being absent for CD34 and CD45 and (3) able to undergo tri-lineage differentiation.
• hMSCs (P2) were seeded in standard 96-well culture plates at cell density of 10 4 cells/ml immersed in 250 ⁇ of culture medium.
• GDF-5 supplemented medium (either 0, 5, 25, 50, 100 or 500 ng/ml) were added to the cultures three days after seeding. Cells were incubated for an additional two days before 25 ⁇ of alamar blue reagent (Invitrogen-Gibco, USA) was added to the medium. Culture plates were protected from light using aluminium foil. Absorbance readings at 570 nm and 600 nm were read using a spectrophotometer (Epoch, Biotek, USA) at 0, 2, 4, 6, 12 and 24, 36, 48 and 60 hours. Untreated hMSCs cultured in MSCs growth medium were used as controls. Dose and Temporal Effects in hMSC Proliferation Using GDF-5 Induction
• hMSCs (P2) were seeded in standard 6- well culture plates at a density of 2 x 10 4 cells per well using serum-free DMEM supplemented with either 0, 5, 25, 50, 100 or 500 ng/ml of recombinant hGDF-5 (R&D Systems, Inc., Minneapolis, MN). Tenocytes isolated as previously described were seeded in similar density to that of hMSCs and were used for comparison. These cells were not supplemented with GDF-5. For dose response analysis, total collagen expressions were measured at 96 hours. Based on the result obtained from this experiment, only three concentrations i.e. 0, 50 and 100 ng/ml of GDF-5 were selected for further analysis which determines the collagen and gene expression levels over time (i.e. day 4, 7 and 10).
• total collagen assays were conducted at day 4, 7, and 10 in hMSCs culture supplemented with 0, 50 and 100 ng/ml of GDF-5.
• Total collagen expression in cultured tenogenic-hMSC was quantified using SircolTM soluble collagen assay kit (Biocolor, Ireland).
• Cell culture medium mixed with 1ml of Sircol dye reagent was agitated vigorously in a 1.5 ml microcentrifuge tube for 30 minutes. The mixtures were then centrifuged for 10 minutes at 10,000xg to collect the collagen-dye complex at the bottom of the centrifuge tubes. The unbound dye solutions were later removed by draining the tubes. Subsequently, 1ml of the alkaline reagent was added to each microcentrifuge tube and mixed.
• the dose response of hMSCs to GDF-5 in their total collagen expression was determined using different amounts of GDF-5 in cell culture medium.
• the results of total collagen assay revealed that at 100 ng/ml, GDF-5 induce significant elevated response (9.983 ⁇ 1.695 ⁇ g/ml: p ⁇ 0.05, Table 1) as compared to 0, 5 and 25 ng/ml of GDF-5 ( Figure 2a).
• This expression level was comparable to that observed in tenocyte culture (10.387 ⁇ 2.316 ⁇ g/ml). No significant differences were observed in the collagen concentration between cultures supplemented with 50, 100 or 500 ng/ml of GDF-5 (Table 1).
• Table 1 Statistical analysis of total collagen expression in hMSCs culture medium at different concentration of GDF-5 at 96 hours of cell culture. Summary of least significant differences (LSD) analysis with Bonferroni adjustment for multiple pairwise comparisons of mean total collagen differences in the culture medium of hMSCs supplemented with different amount of GDF-5. The p- value was presented at 95% confidence interval and significant value was denoted with an asterisk (** Highly significant difference (1% ), * Significant difference (5%).
• hMSCs were cultured in DMEM supplemented with GDF-5 at 0, 50 and lOOng/ml. After 4 days, the degree of cell differentiation was determined by using real-time PCR (qPCR). This was achieved by measuring scleraxis (Sex), type-I collagen (Col-I), type-Ill collagen (Col- III), decorin (Dec) and nucleostemin (Nsf) gene expressions.
• qPCR real-time PCR
• Quantitative polymerase chain reaction was performed using a Bio-Rad CFX96TM Realtime detection system (Bio-Rad Laboratories, Inc., Hercules, CA) in a final volume of 20 ⁇ withlO uL iQ SYBR® Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA), 0.6 L RT samples and 0.2 ⁇ of each primer (for type-I collagen (Col-/), type- III collagen (Col-III), scleraxis (Sex), decorin (Dec), nucleostemin (Nst); Supplementary Material #1).
• qPCR Quantitative polymerase chain reaction
• the amplification protocol was as follows: an initial denaturation and activation step at 95°C for 30s followed by 40 cycles of 95°C for 15s and 61°C for 45s.
• a melting curve program was carried out routinely to confirm the presence of a single product (55-95°C with a heating rate of 0.5°C per second and a continuous fluorescence measurement).
• the annealing temperature at 61°C was derived empirically using temperature gradients.
• To estimate amplification efficiency a standard curve was generated for each target molecule via 5-fold serial dilution of a cDNA pool containing the target gene sequences. Data was analyzed using the CFX manager software. A relative quantification method (with corrected PCR efficiency (Pfaffl, 2001)) was performed.

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• 2013
  • 2013-06-13 WO PCT/MY2013/000108 patent/WO2013176538A2/fr not_active Ceased

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