Enhanced msc preparations

Abstract

The present invention provides preparations of MSCs with important therapeutic potential. The MSC cells are non-primary cells with an antigen profile comprising less than about 1.25% CD45+ cells (or less than about 0.75% CD45+), at least about 95% CD105+ cells, and at least about 95% CD166+ cells. Optionally, MSCs of the present preparations are isogenic and can be expanded ex vivo and cryopreserved and thawed, yet maintain a stable and uniform phenotype. Methods are taught here of expanding these MSCs to produce a clinical scale therapeutic preparations and medical uses thereof.

1 . A mesencymal stem cell (MSC) preparation comprising:
 a. at least about 1 billion human bone marrow-derived MSCs; and   b. an antigen profile comprising:
 i. less than about 0.75% CD45+ cells; 
 ii. at least about 95% CD105+ cells; and 
 iii. at least about 95% CD166+ cells. 
   
     
     
         2 . The MSC preparation of  claim 1 , wherein the at least about 1 billion human bone marrow-derived MSCs are isogenic. 
     
     
         3 . The MSC preparation  claim 2 , wherein the MSC preparation comprises at least about 20 billion human bone marrow-derived MSCs in number. 
     
     
         4 . The MSC preparation  claim 1 , wherein the MSCs express at least about 13 pg to about 44 pg TNFRI per million MSCs and optionally wherein the MSCs, when mixed with peripheral blood mononuclear cells (PBMCs) at a ratio of about 5 PBMCs per MSC, are capable of inhibiting IL2Rα expression by CD3/CD28-activated PBMCs cells by at least about 30%, relative to a control. 
     
     
         5 . The MSC preparation of  claim 4 , wherein the MSCs are isogenic and are at least 4.5×10 9  human bone marrow-derived MSCs in number. 
     
     
         6 . The MSC preparation of  claim 5 , further comprising a cryopreservative. 
     
     
         7 . The MSC preparation of  claim 6 , wherein after a freeze-thaw cycle, at least about 70% of the MCSs are viable, as assessed by dye exclusion. 
     
     
         8 . The MSC preparation of  claim 5 , wherein the MSCs:
 a. are capable of at least 1 population doubling; and   b. retain said antigen profile after the population doubling.   
     
     
         9 . The MSC preparation of  claim 5 , wherein the MSCs:
 a. are capable of at least 1 population doubling; and retain the differentiation capacity after the population doubling.   
     
     
         10 . The MSC preparation of  claim 1 , wherein the preparation contains less than 55 μg/mL BSA and less than 42 μg/mL trypsin. 
     
     
         11 . The MSC preparation of  claim 1 , wherein the preparation is substantially free of:
 a. mycoplasma, endotoxin, and fungi; and   b. viral nucleic acids, wherein viral nucleic acids comprise HTLV1, HTLV2, HBV, CMV, EBV, HHV-6A, HHV-6B, HHV-8, HIV, Parvovirus B-19, HCV, and HPV Type 18 nucleic acids.   
     
     
         12 . A method of making an MSC preparation comprising the steps of:
 a. making a plurality of preparations, each preparation made by the steps comprising:
 i. obtaining from a single donor at least about 70 ml of non-fetal human bone marrow aspirate containing viable cells; 
 ii. expanding by passage expansion the number of viable cells to provide a preparation of at least about 1 billion of the viable cells, wherein the passage expansion comprises establishing a primary culture of isolated MSCs and then serially establishing at least a first non-primary (P1) culture of isolated MSCs from the previous culture; and 
   b. selecting from the plurality of preparations at least one preparation that has an antigen profile comprising:
 i. less than about 0.75% CD45+ cells; 
 ii. at least about 95% CD105+ cells; and 
 iii. at least about 95% CD166+ cells; and 
   c. optionally, wherein the step of selecting comprises testing the at least one preparation to determine the percent CD45+ cells, the percent CD1055+ cells, the percent CD166+ cells.   
     
     
         13 . The method of making an MSC preparation of  claim 12 , wherein the step of expanding comprises expanding the number of cells by at least about 20 population doublings. 
     
     
         14 . The method of making an MSC preparation of  claim 13 , wherein the step of expanding comprises expanding the number of cells by less than about 30 population doublings. 
     
     
         15 . The method of making an MSC preparation of  claim 13 , wherein the step of expanding comprises producing a preparation of at least about 20 billion cells. 
     
     
         16 . The method of making an MSC preparation of  claim 12 , further comprising a step of cryogenically preserving the preparation, optionally wherein at least about 70% of the MSCs are viable after thawing. 
     
     
         17 . The method of making a MSC preparation of  claim 12 , wherein the step of establishing a primary culture of isolated MSCs comprises:
 a. isolating an MSC-containing population from the bone marrow aspirate, optionally wherein the MSC-containing population is a population of isolated nucleated bone marrow cells (INBMCs);   b. seeding the MSC-containing population on a substrate at a concentration in a range of about 50 to about 1000 cells per cm 2  or 146±about 100 cells per cm 2 ; and   c. culturing the MSC-containing population for a period of time.   
     
     
         18 . The method of making a MSC preparation of  claim 12 , wherein the step of serially establishing at least a first non-primary (P1) culture of isolated MSCs from the previous culture comprises:
 a. seeding isolated MSCs from the previously established culture on a substrate in a range of about 1,000 to about 10,000 cells per cm 2  or at about 5,900±about 20% cells per cm 2 ; and then   b. culturing the isolated MSCs for a period of time.   
     
     
         19 . The method of making a MSC preparation of  claim 12 , wherein the step of serially establishing at least a first non-primary (P1) culture of isolated MSCs from the previous culture comprises:
 a. splitting the previous culture at a ratio of about 1:4 to about 1:8 into a plurality of cultures;   b. culturing the plurality of cultures for a period of time; and then   c. optionally, pooling the plurality of cultures.   
     
     
         20 . A method of treating a human in need thereof comprising the step of administering a therapeutic dose of the MSCs from a preparation of  claim 1 .