Perinatal tissue derived mesenchymal stem cells: method of preparation and uses thereof
The present invention discloses the preparation of placenta tissue derived CD106 high CD151+Nestin+ mesenchymal stem cells (MSCs). In a first aspect, the invention relates to a particular method to prepare these cells at industrial scale and the cell population generated thereby. In a second aspect, the invention relates to a cell culture obtained by said particular method, containing placental CD106 high CD151+Nestin+ MSCs expressing the vascular cell adhesion molecule 1 (VCAM-1) marker. The present application shows that said placental CD106 high CD151+Nestin+ MSCs are capable of inducing angiogenesis in vitro and in vivo. The herein presented results also show that administering said placental CD106 high CD151+Nestin+ MSCs to individuals suffering from an ischemic disease or from a disorder of the circulatory system results in a detectable improvement of one or more symptoms of said disease or disorder. Therefore, in a third aspect, the invention relates to placental CD 106 high CD151+Nestin+ MSCs for use as a medicament for treating subjects suffering from an ischemic disease, a disorder of the circulatory system, an immune disease, an organ injury or an organ function failure.
1. A method for obtaining CD106+CD151+Nestin+mesenchymal stem cells (MSCs), said method comprising:
culturing a population of undifferentiated MSCs in a first culture medium devoid of any exogenous growth factor until said undifferentiated MSCs reach 85-90% confluence,
removing the first culture medium,
introducing a second culture medium to the culture of undifferentiated MSCs comprising between 10 and 20 ng/ml of added Interleukin 1β and between 10 and 20 ng/ml of added Interleukin 4,
culturing said undifferentiated MSCs in the second culture medium for at least 48 hours to obtain CD106+CD151+Nestin+MSCs, and
characterizing the CD106+CD151+Nestin+MSCs based on marker expression,
wherein over 60% of the CD106+CD151+Nestin+MSCs express CD106 at a detectible level,
wherein over 98% of the CD106+CD151+Nestin+MSCs express CD151 at a detectible level,
wherein over 98% of the CD106+CD151+Nestin+MSCs express Nestin at a detectible level
wherein over 95% of the CD106+CD151+Nestin+MSCs express CD73, CD90, CD105, and CD166 at a detectible level, and
wherein less than 2% of the CD106+CD151+Nestin+MSCs express CD45, CD34, and HLA-DR at a detectible level.
2. The method of claim 1 , wherein said undifferentiated MSCs are obtained by cell isolation from an explant of umbilical cord fragment or by cell isolation from a placenta tissue fragment.
3. The method of claim 1 , wherein over 98% of the MSCs do not express markers CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, and CD133 at a detectable level.
4. A method for enhancing the CD106 expression level of undifferentiated CD106+CD151+Nestin+mesenchymal stem cells (MSCs), said method comprising:
culturing a population of undifferentiated MSCs in a first culture medium devoid of any exogenous growth factors until said undifferentiated MSCs reach 85-90% confluence,
removing the first culture medium,
introducing a second culture medium to the culture of undifferentiated MSCs comprising between 10 and 20 ng/ml of added Interleukin 1β and between 10 and 20 ng/ml of added Interleukin 4,
culturing said undifferentiated MSCs in the second culture medium for at least 48 hours to produce undifferentiated CD106+CD151+Nestin+MSCs, and
characterizing the CD106+CD151+Nestin+MSCs based on marker expression,
wherein the CD106 expression is enhanced such that over 60% of the undifferentiated CD106+CD151+Nestin+MSCs express CD106 at a detectible level,
wherein over 98% of the undifferentiated CD106+CD151+Nestin+MSCs express CD151 at a detectible level,
wherein over 98% of the undifferentiated CD106+CD151+Nestin+MSCs express Nestin at a detectible level,
wherein over 95% of the undifferentiated CD106+CD151+Nestin+MSCs express CD73, CD90, CD105, and CD166 at a detectible level, and
wherein less than 2% of the undifferentiated CD106+CD151+Nestin+MSCs express CD45, CD34, and HLA-DR at a detectible level.
5. The method of claim 4 , wherein said undifferentiated MSCs are obtained by cell isolation from a placenta tissue fragment.
6. The method of claim 4 , wherein over 98% of the MSCs do not express markers CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, and CD133 at a detectable level.
7. A method for obtaining CD106+CD151+Nestin+mesenchymal stem cells (MSCs), comprising the steps of:
a) collecting mononuclear cells contained in a perinatal biological tissue or fluid,
b) culturing the mononuclear cells in a first culture medium devoid of any exogenous growth factor,
wherein said mononuclear cells are passaged when they reach 85-90% confluence,
c) characterizing the mononuclear cells based on marker expression to obtain a population of undifferentiated MSCs,
wherein 95% of the undifferentiated MSCs express the markers CD73, CD90, CD105 and CD166, and less than 2% express the markers CD45, CD34 and HLA-DR,
d) seeding the population of undifferentiated MSCs obtained from step c) at a density of 1000 to 5000 MSCs per cm2 into a second culture medium,
e) once the population of undifferentiated MSCs in step d) reaches 40-50% confluency, adding between 10 and 20 ng/ml of Interleukin 1β and between 10 and 20 ng/ml of Interleukin 4, and culturing said population of undifferentiated MSCs for at least 48 hours,
f) collecting the population of undifferentiated MSCs from step e) when they reach 90-95% confluence,
g) characterizing the population of undifferentiated MSCs obtained from step f) based on expression of markers comprising CD106, CD151, and Nestin to obtain a population of CD106+CD151+Nestin+MSCs,
wherein the CD106 expression is enhanced such that over 60% of the undifferentiated CD106+CD151+Nestin+MSCs express CD106 at a detectible level,
wherein over 98% of the undifferentiated CD106+CD151+Nestin+MSCs express CD151 at a detectible level,
wherein over 98% of the undifferentiated CD106+CD151+Nestin+MSCs express Nestin at a detectible level.
8. The method of claim 7 , wherein over 98% of the MSCs do not express markers CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104, and CD133 at a detectable level.