Methods for differentiating mesenchymal stem cells

Abstract

The application discloses a method for obtaining MSC-derived cells with improved transplantation properties from MSC, the method comprising a cell size reduction step, wherein said cell size reduction step is characterized by contacting MSC or MSC-derived cells in vitro or ex vivo with heparin or a derivative or analogue thereof at a concentration of at least 0.01 IU/ml. The application further provides a method for obtaining mesenchymal stem cell-derived cells from mesenchymal stem cells (MSC) comprising contacting MSC in vitro or ex vivo with FGF-2, TGFβ and at least 0.01 IU/ml heparin or a derivative or analogue thereof. The invention also provides the so-obtained cells and cell populations, as well as further products comprising such and uses thereof.

1 . A method for obtaining progenitor cells committed to the osteochondroblastic lineage from mesenchymal stem cells (MSC), the method comprising:
 differentiating MSC into progenitor cells committed to the osteochondroblastic lineage by contacting the MSC in vitro or ex vivo with
 fibroblast growth factor-2 (FGF-2), 
 transforming growth factor beta (TGFβ), and 
 heparin or a derivative or analogue thereof at a concentration of at least 0.01 IU/ml; and 
 harvesting the progenitor cells committed to the osteochondroblastic lineage so as to obtain a substantially pure population of the progenitor cells committed to the osteochondroblastic lineage; 
   wherein the progenitor cells committed to the osteochondroblastic lineage have the ability to differentiate into cells of the osteoblastic lineage and/or into cells of the chondroblastic lineage.   
     
     
         2 . The method according to  claim 1 ,
 wherein the MSC are MSC recovered from a biological sample of a subject and   wherein, after contacting the MSC with FGF-2, TGFβ, and heparin or derivative or analogue thereof,   removing non-adherent matter; and   culturing adherent cells in the medium comprising FGF-2, TGFβ, and heparin or a derivative or analogue thereof at a concentration of at least 0.01 IU/ml,   thereby obtaining the progenitor cells committed to the osteochondroblastic lineage.   
     
     
         3 . The method according to  claim 1 , wherein TGFβ is selected from the group consisting of TGFβ1, TGFβ2, TGFβ3, and mixtures thereof. 
     
     
         4 . A method for obtaining progenitor cells committed to the osteochondroblastic lineage with improved transplantation properties from mesenchymal stem cells (MSC), the method comprising:
 differentiating the MSC into progenitor cells committed to the osteochondroblastic linage by a size reduction step, wherein the size reduction step comprises contacting the MSC or progenitor cells committed to the osteochondroblastic lineage in vitro or ex vivo with heparin or a derivative or analogue thereof at a concentration of at least 0.01 IU/ml; and   harvesting the progenitor cells committed to the osteochondroblastic lineage so as to obtain a substantially pure population of the progenitor cells committed to the osteochondroblastic lineage;   wherein the progenitor cells committed to the osteochondroblastic lineage have the ability to differentiate into cells of the osteoblastic lineage and/or into cells of the chondroblastic lineage.   
     
     
         5 . The method according to  claim 1 , wherein:
 the concentration of heparin or derivative or analogue thereof is at least 0.05 IU/ml; and/or
 the heparin or heparin derivative or analogue thereof is selected from the group consisting of unfractionated heparin (UFH), low molecular weight heparin (LMWH), a heparin salt, a heparinoid salt, a heparin fragment, a heparinoid fragment, and mixtures thereof. 
   
     
     
         6 . The method according to  claim 1 , wherein at least 60% of the progenitor cells committed to the osteochondroblastic lineage have a diameter equal to or less than 25 μm (D 60 ≤25 μm) in suspension and wherein at most 5% of the progenitor cells committed to the osteochondroblastic lineage have a diameter of more than 35 μm in suspension. 
     
     
         7 . The method according to  claim 1 , wherein the MSC or progenitor cells committed to the osteochondroblastic lineage are additionally contacted with one or more of plasma or serum. 
     
     
         8 . A method for obtaining progenitor cells committed to the osteochondroblastic lineage from mesenchymal stem cells (MSC), the method comprising:
 differentiating MSC into progenitor cells committed to the osteochondroblastic lineage by contacting the MSC in vitro or ex vivo with FGF-2, TGFβ, and heparin or a derivative or analogue thereof,   wherein at least 60% of the progenitor cells committed to the osteochondroblastic lineage have a diameter equal to or less than 25 μm (D 60 ≤25 μm) in suspension and wherein at most 5% of the progenitor cells committed to the osteochondroblastic lineage have a diameter of more than 35 μm in suspension; and
 harvesting the progenitor cells committed to the osteochondroblastic lineage so as to obtain a substantially pure population of the progenitor cells committed to the osteochondroblastic lineage; 
   wherein the progenitor cells committed to the osteochondroblastic lineage have the ability to differentiate into cells of the osteoblastic lineage and/or into cells of the chondroblastic lineage.   
     
     
         9 . The method according to  claim 8 , wherein:
 MSC are contacted with heparin or derivative or analogue thereof at a concentration of at least 0.01 IU/ml; and/or
 TGFβ is selected from the group consisting of TGFβ1, TGFβ2, TGFβ3, and mixtures thereof. 
   
     
     
         10 . The method according to  claim 5 , wherein the concentration of heparin or derivative or analogue thereof is about 0.1 IU/ml. 
     
     
         11 . The method according to  claim 3 , wherein TGFβ is TGFβ1. 
     
     
         12 . The method according to  claim 9 , wherein TGFβ is TGFβ1. 
     
     
         13 . The method according to  claim 1 , wherein the progenitor cells committed to the osteochondroblastic lineage are osteochondroprogenitors, osteoprogenitors, pre-osteoblasts, chondroprogenitors and/or pre-chondroblasts. 
     
     
         14 . The method according to  claim 1 , wherein the substantially pure population of progenitor cells committed to the osteochondroblastic lineage comprises at least 90% by number progenitor cells committed to the osteochondroblastic lineage. 
     
     
         15 . The method according to  claim 1 , wherein the substantially pure population of progenitor cells committed to the osteochondroblastic lineage comprises at least 95% by number progenitor cells committed to the osteochondroblastic lineage. 
     
     
         16 . The method according to  claim 1 , wherein the the substantially pure population of progenitor cells committed to the osteochondroblastic lineage comprises progenitor cells committed to the osteochondroblastic lineage at a concentration between about 1×10 7 /ml and about 1×10 8 /ml of the population. 
     
     
         17 . The method according to  claim 1 , wherein the progenitor cells committed to the osteochondroblastic lineage comprise one or more of:
 increased expression of a gene encoding an osteoblastic marker selected from the group consisting of Runt-related transcription factor 2 (RUNX2), alkaline phosphatase, biomineralization associated (ALPL), bone morphogenetic protein 2 (BMP2), osteoprotegerin (OPG), periostin (POSTN), cell adhesion molecule 1 (CADM1), connexin 43 (CX43), membrane metalloendopeptidase (CD10), and WNT1 inducible signaling pathway 1 (WISP1) as compared to the expression of the respective gene in MSC; and   increased expression of a gene encoding a chondroblastic marker selected from the group consisting of SRY-box transcription factor 9 (SOX9), zinc finger protein 521 (ZNF521), chitinase 3 like 1 (CH13L1), and matrix metallopeptidase 13 (MMP13) as compared to the expression of the respective gene in MSC.   
     
     
         18 . The method according to  claim 1 , wherein the progenitor cells committed to the osteochondroblastic lineage comprise increased expression of a gene encoding an osteoblastic marker selected from the group consisting of Runt-related transcription factor 2 (RUNX2), alkaline phosphatase, biomineralization associated (ALPL), bone morphogenetic protein 2 (BMP2), osteoprotegerin (OPG), periostin (POSTN), cell adhesion molecule 1 (CADM1), connexin 43 (CX43), membrane metalloendopeptidase (CD10), and WNT1 inducible signaling pathway 1 (WISP1) as compared to the expression of the respective gene in MSC.