WO2013176538A2 - Milieu ténogène de différenciation de cellules souches mésenchymateuses dans une lignée ténogène - Google Patents

Milieu ténogène de différenciation de cellules souches mésenchymateuses dans une lignée ténogène Download PDF

Info

Publication number
WO2013176538A2
WO2013176538A2 PCT/MY2013/000108 MY2013000108W WO2013176538A2 WO 2013176538 A2 WO2013176538 A2 WO 2013176538A2 MY 2013000108 W MY2013000108 W MY 2013000108W WO 2013176538 A2 WO2013176538 A2 WO 2013176538A2
Authority
WO
WIPO (PCT)
Prior art keywords
tenogenic
mesenchymal stem
gdf
stem cells
cells
Prior art date
Application number
PCT/MY2013/000108
Other languages
English (en)
Other versions
WO2013176538A3 (fr
Inventor
Tunku Kamarul Zaman Tunku Zainol Abidin MADYA
Sik Loo TAN
Original Assignee
University Of Malaya
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Malaya filed Critical University Of Malaya
Publication of WO2013176538A2 publication Critical patent/WO2013176538A2/fr
Publication of WO2013176538A3 publication Critical patent/WO2013176538A3/fr

Links

Classifications

    • 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.

Abstract

Les ténocytes sont des cellules hautement différenciées qui présentent un potentiel de réplication limité. Par conséquent, une propagation in vitro de ténocytes peut ne pas fournir encore suffisamment de cellules ensemencées pour une ingénierie tissulaire du tendon. Des cellules souches mésenchymateuses (MSC) de moelle osseuse sont aptes à s'autorenouveler et sont adaptées à se différencier en une lignée multiple comprenant des ténocytes. Il est par conséquent possible que des MSC puissent être utilisées en tant que cellules ensemencées dans l'ingénierie tissulaire du tendon. La présente invention concerne ainsi un procédé et un milieu de culture permettant de favoriser la différenciation de cellules souches mésenchymateuses (MSC) de moelle osseuse humaines dans une lignée ténogène.
PCT/MY2013/000108 2012-05-22 2013-06-13 Milieu ténogène de différenciation de cellules souches mésenchymateuses dans une lignée ténogène WO2013176538A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
MYPI2012002249 2012-05-22
MYPI2012002249 2012-05-22

Publications (2)

Publication Number Publication Date
WO2013176538A2 true WO2013176538A2 (fr) 2013-11-28
WO2013176538A3 WO2013176538A3 (fr) 2014-03-06

Family

ID=49624465

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/MY2013/000108 WO2013176538A2 (fr) 2012-05-22 2013-06-13 Milieu ténogène de différenciation de cellules souches mésenchymateuses dans une lignée ténogène

Country Status (1)

Country Link
WO (1) WO2013176538A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110295140A (zh) * 2019-06-04 2019-10-01 河北贝特赛奥生物科技有限公司 一种无血清培养骨髓间充质干细胞的方法
EP3872168A1 (fr) 2020-02-28 2021-09-01 Cline Scientific AB Différenciation de chondrocytes
WO2024000073A1 (fr) * 2022-06-30 2024-01-04 Stemcell Technologies Canada Inc. Milieux, kits et procédés de différenciation de ténocytes ou de chondrocytes

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130122589A1 (en) * 2010-07-26 2013-05-16 The University Of Manchester Targeted differentiation of stem cells

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110295140A (zh) * 2019-06-04 2019-10-01 河北贝特赛奥生物科技有限公司 一种无血清培养骨髓间充质干细胞的方法
EP3872168A1 (fr) 2020-02-28 2021-09-01 Cline Scientific AB Différenciation de chondrocytes
WO2021173066A1 (fr) 2020-02-28 2021-09-02 Cline Scientific Ab Procédé de préparation d'un implant cartilagineux à chondrocytes
WO2024000073A1 (fr) * 2022-06-30 2024-01-04 Stemcell Technologies Canada Inc. Milieux, kits et procédés de différenciation de ténocytes ou de chondrocytes

Also Published As

Publication number Publication date
WO2013176538A3 (fr) 2014-03-06

Similar Documents

Publication Publication Date Title
Zhang et al. MicroRNA-30b is a multifunctional regulator of aortic valve interstitial cells
Zhang et al. A signal-amplification circuit between miR-218 and Wnt/β-catenin signal promotes human adipose tissue-derived stem cells osteogenic differentiation
Byun et al. FGF2 stimulates osteogenic differentiation through ERK induced TAZ expression
Heo et al. Wnt/β-catenin signaling enhances osteoblastogenic differentiation from human periodontal ligament fibroblasts
Van Dijk et al. Differentiation of human adipose-derived stem cells towards cardiomyocytes is facilitated by laminin
Chen et al. Inhibiting actin depolymerization enhances osteoblast differentiation and bone formation in human stromal stem cells
Xiao et al. MicroRNA-204 is required for differentiation of human-derived cardiomyocyte progenitor cells
Wang et al. RNA-seq based transcriptome analysis of endothelial differentiation of bone marrow mesenchymal stem cells
Zhao et al. Age-related changes in nucleus pulposus mesenchymal stem cells: an in vitro study in rats
Ji et al. Transdifferentiation of human endothelial progenitors into smooth muscle cells
He et al. Pulsed electromagnetic fields inhibit human osteoclast formation and gene expression via osteoblasts
US20230174929A1 (en) Method for generating mesoderm and/or endothelial colony forming cell-like cells having in vivo blood vessel forming capacity
WO2015042356A1 (fr) Milieu de culture chimiquement défini pour le maintien et la différenciation de cellules souches
Humenik et al. Canine Bone Marrow-derived Mesenchymal Stem Cells: Genomics, Proteomics and Functional Analyses of Paracrine Factors*[S]
Xue et al. Presence of an established calcification marker in trabecular meshwork tissue of glaucoma donors
Kalomoiris et al. Fibroblast Growth Factor 2 Regulates High Mobility Group A2 Expression in Human Bone Marrow‐Derived Mesenchymal Stem Cells
Igarashi et al. Selection of common markers for bone marrow stromal cells from various bones using real-time RT-PCR: effects of passage number and donor age
Riha et al. Cyclic strain induces vascular smooth muscle cell differentiation from murine embryonic mesenchymal progenitor cells
KR101562366B1 (ko) 메타크릴레이트화된 젤라틴을 포함하는, 심근세포분화 유도용 세포 지지체
Zhao et al. MicroRNA-1 effectively induces differentiation of myocardial cells from mouse bone marrow mesenchymal stem cells
Rui et al. CTGF attenuates tendon-derived stem/progenitor cell aging
Kishimoto et al. Fad104, a positive regulator of adipogenesis, negatively regulates osteoblast differentiation
Zhang et al. Downregulation of microRNA‑143 promotes osteogenic differentiation of human adipose‑derived mesenchymal stem cells through the k‑Ras/MEK/ERK signaling pathway
WO2013176538A2 (fr) Milieu ténogène de différenciation de cellules souches mésenchymateuses dans une lignée ténogène
Qian et al. Novel miR-sc4 regulates the proliferation and migration of Schwann cells by targeting Cdk5r1

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13793201

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 13793201

Country of ref document: EP

Kind code of ref document: A2