Growth of neural precursor cells using umbilical cord blood serum and a process for the preparation thereof for therapeutic purposes

Abstract

This invention is concerned with stem cells derived from umbilical cord blood serum and a method for growing human embryonic stem cells and adult cells comprising sera separated from clotted umbilical cord blood, including growing and differentiating cord blood stem cells into neural precursors, comprising transdifferentiating CD34+ stem cells from mononuclear cells derived from umbilical cord blood to neural precursors. The stem cells obtained from the umbilical cord include pluripotent stem and progenitor cell population of mononuclear cells, and separating pluripotent stem and progenitor cell population of mononuclear cells obtained from the umbilical cord blood. A magnetic cell separator is used to separate out cells which contain a CD marker and then expanding the cells in a growth medium containing retinoic acid and one or more growth factors BDNF, GDNF, NGF and FGF as a differentiating agent. The invention is also concerned with the transplantation and repair of nerve damage, stokes, spinal injury, Parkinson's and Alzheimer's, prepared in accordance with the aforesaid method and a media for culturing umbilical cord blood stem calls consisting essentially of cord blood stem cells derived from umbilical cord blood serum.

1 . A method for growing human embryonic stem cells and adult cells comprising sera separated from clotted umbilical cord blood.  
     
     
         2 . The method as claimed in  claim 1 , wherein the adult cells are neural precursor cells.  
     
     
         3 . The method as claimed in  claim 1  including transdifferentiation of CD+ 34 stem cells from mononuclear cells derived from the umbilical cord blood.  
     
     
         4 . The method as claimed in  claim 1  for growing and differentiating cord blood stem cells into neural precursors, comprising transdifferentiating CD34+ stem cells from mononuclear cells derived from umbilical cord blood to neural precursors.  
     
     
         5 . The method according to  claim 1  comprising growing and differentiating cord blood stem cells into neutral precursors.  
     
     
         6 . The method as claimed in  claim 5  wherein the stem cells obtained from the umbilical cord include pluripotent stem and progenitor cell population of mononuclear cells, and including the step of separating pluripotent stem and progenitor cell population of mononuclear cells obtained from the umbilical cord blood.  
     
     
         7 . The method as claimed in  claim 1  including a magnetic cell separator to separate out cells which contain a CD marker and then expanding these cells in a growth medium containing retinoic acid and one or more growth factors selected from the group consisting of BDNF, GDNF, NGF and FGF and mixtures thereof as a differentiating agent.  
     
     
         8 . The method according to  claim 7  wherein the differentiating agent is retinoic acid  
     
     
         9 . The method according to  claim 1  including a differentiating agent selected from the group consisting of 9-cis retinoic acid, all-trans retinoic and combinations thereof.  
     
     
         10 . The method according to  claim 9  including growth factor selected from the group consisting of BDNF, GDNF, NGF or FGF and combinations thereof.  
     
     
         11 . The method according to  claim 6  wherein pluripotent and progenitor cells are obtained from the group selected from fresh umbilical cord blood, reconstituted cryopreserved umbilical cord blood and a combination of a fresh or reconstituted cryopreserved mononuclear cell fraction thereof.  
     
     
         12 . The method of growing mononuclear cells in a standard cellular media, comprising the steps of growing the cells in a neural proliferation medium, and then growing the cells in a differentiation medium.  
     
     
         13 . The method according to  claim 13 , wherein the standard cellular media is a medium supplemented with non-essential amino acids, glutamine acid serum.  
     
     
         14 . The method according to  claim 12  wherein the growth factors are limited or removed.  
     
     
         15 . The method according to  claim 12  wherein the differentiation medium is similar to a neural proliferation medium containing DMEM/F12 1:1 cell medium supplementated with glutamine 2 mM, sodium bicarbonate 3 mM, EGF 20 ng/ml, bFGF10 ng/ml and NGF 100 ng/ml, the neural proliferation medium having neural differentiation agents added.  
     
     
         16 . The method according to  claim 15  including using a magnetic cell separator (MACS) for removing cells which contain a CD marker, and then expanding the remaining cells in a growth medium or differentiating the remaining cells in a growth medium having a differentiating agent, and co-culturing with adult mesenchymal stem cells or accessory cells from other portions of fetal and mature central nervous system.  
     
     
         17 . The method according to  claim 12  including enriching mononuclear cells comprising depleting red blood cell using 3% v/v Dextran (high molecular weight) in the ratio of 1:1 with respect to the volume of blood, collecting and centrifuging Leucocyte rich plasma, washing and layering the cells of Histopaque™ 1077, centrifuging tubes containing the sample at 400 g for 30 minutes, washing and separating mononuclear cells separated from the interface using pipettes, and washed mononuclear cells are then suspended neural proliferation medium.  
     
     
         18 . The method according to  claim 12 , including straining the cells with fluorochrome conjugated antibodies for flow cytometry, removing specified numbers of cells in polystyrene round bottom tubes, staining the anti-CD34-FITC, anti-CD133-PE and anti-CD45-PerCP antibodies, and analyzing the stained cells on a FACSCalibur flow cytometer.  
     
     
         19 . The method as claimed in  claim 12  including plating mononuclear cells from cord blood in Nunc T75 culture flasks in a neural cells proliferation medium containing DMEM/F123 (1:1) supplemented with FBS or CBS, seeding the cells at a density of 1×10 6  to cells/ml, harvesting the non-adherent a fixed culture period of 1 week, counting and analyzing the non-adherent cells for the expression of CD133, CD45 and CD34 markers, whereby the proliferation kinetics of these cells in this medium shows no significant difference (p>0.3) as measured by the paired T test in the numbers of non adherent cells, as observed in cells cultured in the presence of CBS or FBS, thereby including that cord blood serum supports growth of non adherent cells in said cultures with equal efficacy as compared to fetal bovine serum.  
     
     
         20 . The method as claimed in  claim 12 , including plating mononuclear cells from umbilical cord blood in neural cell proliferation medium, feeding the cultures every 3-4 days and allowing the cultures to proceed to 90% confluency, detaching the adherent cells using cell dissociation buffer and seeding them into the next passage with cell dissociation buffer in both these cultures.  
     
     
         21 . The method as claimed in  claim 19 , including using cultures having FBS or CBS.  
     
     
         22 . The method as claimed in claim  26 , including using trypsin-EDTA for detachment of the cells.  
     
     
         23 . Stem cells derived from umbilical cord blood serum for use in connection with transplantation and repair of nerve damage, stokes, spinal injury, Parkinson's and Alzheimer's, prepared in accordance with the method as claimed in  claim 16 .  
     
     
         24 . A media for culturing umbilical cord blood stem calls consisting essentially of cord blood stem cells derived from umbilical cord blood serum.