Generation of Clonal Mesenchymal Progenitors and Mesenchymal Stem Cell Lines Under Serum-Free Conditions

Abstract

Methods for obtaining multipotent mesenchymal stem cells under serum-free conditions and methods for identifying multipotent mesenchymal progenitor cells are disclosed.

1 . A method of generating a clonal population of primate mesenchymal stem cells, the method comprising the steps of:
 culturing a heterogeneous, single-cell suspension of primate cells that contains mesenchymal progenitors in a serum-free, semi-solid medium containing between about  5  and about 100 ng/ml bFGF until independent colonies form; and   culturing one of the independent colonies in a serum-free, liquid medium containing between about 5 and about 100 ng/ml bFGF to obtain an substantially pure clonal population of MSCs.   
     
     
         2 . The method of  claim 1 , wherein the heterogeneous suspension is obtained in a method comprising the steps of:
 co-culturing pluripotent primate cells with bone marrow stromal cells in a medium that supports differentiation for between two and five days until differentiated cells are formed; and   suspending the differentiated cells.   
     
     
         3 . The method of  claim 2 , wherein the pluripotent cells are selected from the group consisting of embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells. 
     
     
         4 . The method of  claim 2 , further comprising the step of depleting cells not derived by in vitro differentiation of pluripotent cells from the heterogeneous suspension. 
     
     
         5 . The method of  claim 4 , wherein the depleting step comprises the steps of:
 non-covalently binding the cells to be depleted to paramagnetic monoclonal antibodies specific for epitopes on the cells to be depleted; and   segregating the antibody-bound cells with a magnet.   
     
     
         6 . The method of  claim 2 , wherein the bone marrow stromal cells are mouse OP9 cells. 
     
     
         7 . The method of  claim 1 , wherein the heterogeneous suspension is obtained in a method comprising the steps of:
 dissociating an embryoid body to single cells; and   suspending the single cells.   
     
     
         8 . The method of  claim 1 , wherein the single-cell suspension is cultured for between ten to twenty days. 
     
     
         9 . The method of  claim 1 , wherein the semi-solid medium contains between about 20 and about 100 ng/ml bFGF. 
     
     
         10 . The method of  claim 1 , wherein the semi-solid medium contains about 5 ng/ml bFGF. 
     
     
         11 . The method of  claim 1 , wherein the semi-solid medium contains about 1% methyl cellulose. 
     
     
         12 . The method of  claim 1 , wherein the semi-solid medium contains between about 10 ng/ml and about 20 ng/ml PDGF-BB. 
     
     
         13 . The method of  claim 1 , wherein cells in the single-cell suspension express MIXL1 and T (BRACHYURY). 
     
     
         14 . The method of  claim 1 , wherein the primate cells are of human origin. 
     
     
         15 . The method of  claim 1 , wherein the MSCs are cultured in the presence of an extracellular matrix protein. 
     
     
         16 . The method of  claim 15 , wherein the extracellular matrix protein is selected from the group consisting of Matrigel®, collagen, gelatin and fibronectin. 
     
     
         17 . The method of  claim 1 , wherein the mesenchymal colonies express FOXF1, MEF2C, MSX1, MSX2, SNAI1, SNAI2, SOX9 and RUNX2. 
     
     
         18 . The method of  claim 1 , wherein the mesenchymal colonies express CD44, CD56 and CD105 expression, but do not express CD31, CD43, CD45 and VE-cadherin. 
     
     
         19 . The method of  claim 1 , wherein the method comprises the step of:
 observing at least one mesenchymal characteristic of colonies formed during culture in the serum-free, semi-solid medium, thereby confirming identification of mesenchymal progenitors in the suspension.   
     
     
         20 . The method of  claim 19  wherein the at least one mesenchymal characteristic is selected from the group consisting of a functional characteristic, a morphological characteristic and a phenotypical characteristic. 
     
     
         21 . The method of  claim 20 , wherein the functional characteristic is selected from the group consisting of (1) growth stimulation by factors that promote mesenchymal cell growth (e.g., PDGF-BB, EGF and TGF-alpha) and growth suppression by factors involved in mesodermal differentiation (e.g., VEGF, TGF-beta and Activin A) and (2) differentiation into osteogenic, chondrogenic or adipogenic cell lineages. 
     
     
         22 . The method of  claim 20 , wherein the morphological characteristic is selected from the group consisting of (1) a tightly packed, round-shaped cell aggregate measuring 100-500 μm in diameter; and (2) lack of dense outer cell layer and irregular inner structure. 
     
     
         23 . The method of  claim 20 , wherein the phenotypical characteristic is selected from the group consisting of (1) expression of CD44, CD56, CD105 and CD140a, but no expression of CD31, CD43, CD45 and VE-cadherin, (2) expression of FOXF1, MEF2C, MSX1, MSX2, SNAI1, SNAI2, SOX9 and RUNX2 and (3) expression of vimentin and alpha smooth muscle actin, but no expression of desmin. 
     
     
         24 . The method of  claim 19 , wherein the method further comprises counting colonies to estimate a number of mesenchymal progenitors in the heterogeneous suspension. 
     
     
         25 . A cell population comprising:
 an substantially pure line of clonally-derived mesenchymal stem cells positive at least for CD44, CD56, CD140a and CD105, but negative for CD31, CD43, CD45 and VE cadherin.   
     
     
         26 . The cell population of  claim 25 , wherein the population comprises at least 99% mesenchymal stem cells.