Stem Cells Culture Systems

Abstract

The present invention concerns systems and methods for providing human cell cultures. Specific embodiments of the invention relate to cultures of feeder cells for use in stem cell technology, as well as cultures, culture systems and methods for maintenance and propagating of stem cells in an undifferentiated state as well as for the development of somatic cells cultures from stem cells, the somatic cell cultures being free of extraembryonic cells.

1 . A cell culture comprising cells obtained from human umbilical cord tissue, the cells obtained from human umbilical cord being capable of maintaining stem cells (SC) in an undifferentiated state when co-cultured therewith. 
   
   
       2 . The cell culture of  claim 1 , wherein the cells obtained from human umbilical cord are feeder cells. 
   
   
       3 . The cell culture of  claim 1 , wherein the lls obtained from human umbilical cord are essentially fibroblast cells. 
   
   
       4 . A culture system for maintenance of stem cells (SC) in an undifferentiated state, the culture system comprising feeder cells expanded from human umbilical cord cells, human embryonic fibroblast cells (HEF) and a combination of same. 
   
   
       5 . The culture system of  claim 4 , wherein said SC are embryonic SC. 
   
   
       6 . The culture system of  claim 4 , wherein said SC are human SC. 
   
   
       7 . The culture system of  claim 4 , wherein said feeder cells comprise fibroblast cells. 
   
   
       8 . The culture system of  claim 4 , for propagation of undifferentiated SC. 
   
   
       9 . The culture system of  claim 4 , being an animal free culture system. 
   
   
       10 . The culture system of  claim 4 , comprising a humanized serum replacement substitute. 
   
   
       11 . The culture system of  claim 4 , being essentially free of antibacterial agents and/or reducing agents. 
   
   
       12 . An undifferentiated pluripotent human embryonic SC culture obtained by incubating a cluster of cells from inside a blastocyst with a culture system according to  claim 4 . 
   
   
       13 . A method for maintaining SC in an undifferentiated state, the method comprising incubating said cells with a culture system comprising feeder cells expanded from human umbilical cord cells, human embryonic fibroblast cells (HEF) or a combination of same. 
   
   
       14 . The method of  claim 13 , wherein said SC are embryonic stem cells. 
   
   
       15 . The method of  claim 13 , wherein said SC are human stem cells. 
   
   
       16 . The method of  claim 13 , wherein said feeder cells comprise fibroblast cells. 
   
   
       17 . The method of  claim 13 , for propagating said SC in an undifferentiated state. 
   
   
       18 . The method of  claim 13 , comprising incubating said SC with a humanized serum replacement substitute. 
   
   
       19 . The method of  claim 13 , wherein said culture system is essentially free of antibacterial agents and/or reducing agents. 
   
   
       20 . A culture system for inhibiting or preventing differentiation of stem cells to extraembryonic cells, the culture system comprising nicotinamide (NA) or a derivative of NA having an inhibitory effect on differentiation of SC to extraembryonic cells similar to that of NA. 
   
   
       21 . The culture system of  claim 20 , where in said SC are embryonic stem cells. 
   
   
       22 . The culture system of  claim 20 , wherein said SC are human stem cells. 
   
   
       23 . The culture system of  claim 20 , capable of inducing differentiation of SC into somatic cells. 
   
   
       24 . The culture system of  claim 23 , for inducing differentiation of said SC to neural precursor cells. 
   
   
       25 . The culture system of  claim 24 , for inducing differentiation of said SC to retinal pigmented epithelial (RPE) cells. 
   
   
       26 . An undifferentiated human embryonic SC culture essentially free of extraembryonic cells, obtained by incubating a cluster of cells from inside a blastocyst with a culture system according to  claim 20 . 
   
   
       27 . A cell culture essentially free of extraembryonic cells and comprising somatic cells obtained by incubating stem cells with a culture system according to  claim 20 . 
   
   
       28 . The cell culture of  claim 27 , wherein said somatic cells are neural precursor cells. 
   
   
       29 . The cell culture of  claim 27 , wherein said somatic cells are retinal pigmented epithelial (RPE) cells. 
   
   
       30 . A method for inhibiting or preventing differentiation of SC to extraembryonic cells, the method comprises incubating said SC in a culture system comprising NA or a derivative of NA having an inhibitory effect on differentiation of SC to extraembryonic cells similar to that of NA. 
   
   
       31 . The method of  claim 30 , wherein said SC are embryonic SC. 
   
   
       32 . The method of  claim 30 , wherein said SC are human SC. 
   
   
       33 . The method of  claim 30 , wherein in the presence of said NA or said derivative of NA, said SC differentiate to somatic cells. 
   
   
       34 . The method of  claim 33 , wherein said somatic cells are neural precursor cells. 
   
   
       35 . The method of  claim 33 , wherein said somatic cells are retinal pigmented epithelial (RPE) cells. 
   
   
       36 . The method of  claim 30 , comprising incubating said SC in a culture system comprising between about 1 mM to about 20 mM NA or a NA derivative having an inhibitory effect on differentiation of SC to extraembryonic SC similar to that of NA. 
   
   
       37 . The method of  claim 30 , comprising culturing said SC in a suspension. 
   
   
       38 . The method of  claim 30 , for obtaining survival of said SC in said culture system for at least 12 weeks. 
   
   
       39 . The method of  claim 30 , wherein said NA induces an increase in number of cells within embryoid bodies (EB). 
   
   
       40 . A serum free culture system for maintenance of SC in an undifferentiated state, the serum free culture system comprising animal reagent free basic medium, and humanized serum replacement substitute. 
   
   
       41 . The serum free culture system of  claim 40 , being a humanized culture system. 
   
   
       42 . The serum free culture system of  claim 40 , wherein said basic medium is selected from Cellgro Stem Cell Growth Medium, KO DMEM, Neurobasal™, or X-Vivo 10. 
   
   
       43 . The serum free culture system of  claim 40 , wherein said humanized serum replacement substituent is selected from TCH™, Nutridoma-CS, N2 or combination of same. 
   
   
       44 . The serum free culture system of  claim 40 , wherein when said basic medium is Neurobasal™, the system further comprises a N2 supplement or a modification of N2 supplement. 
   
   
       45 . The serum free culture system of  claim 40 , wherein said SC are embryonic SC. 
   
   
       46 . The serum free culture system of  claim 40 , wherein said SC are human SC. 
   
   
       47 . The serum free culture system of  claim 40 , comprising human derived feeder cells. 
   
   
       48 . The serum free culture system of  claim 47 , wherein the feeder cells comprise fibroblast cells. 
   
   
       49 . The serum free culture systems of  claim 48 , wherein said fibroblast cells are selected from human embryonic fibroblast cells (HEF), umbilical cord derived fibroblast cells and foreskin derived fibroblast cells. 
   
   
       50 . An undifferentiated human embryonic SC culture obtained by incubating a cluster of cells from inside a blastocyst with a serum free culture system according to  claim 40 . 
   
   
       51 . A method of maintaining SC in an undifferentiated state, the method comprises incubating said cells with a culture system comprising serum free culture system comprising serum free basic medium and humanized serum replacement substitute. 
   
   
       52 . The method of  claim 51 , wherein said serum free culture system comprises a e basic medium selected from Cellgro Stem Cell Growth Medium, KO DMEM, Neurobasal™, or X-Vivo 10. 
   
   
       53 . The method of  claim 51 , wherein said humanized serum replacement substituent is selected from TCH™, Nutridoma-CS N2 supplement or a modification of N2 supplement or combination of same. 
   
   
       54 . The method of  claim 51 , wherein when said basic medium is Neurobasal™, the system further comprises a N2 supplement or a modification of N2 supplement. 
   
   
       55 . The method of  claim 51 , for maintaining embryonic SC in an undifferentiated state. 
   
   
       56 . The method of  claim 51 , for maintaining human SC. 
   
   
       57 . The method of  claim 51 , comprising incubating said cells with human derived feeder cells. 
   
   
       58 . The method of  claim 57 , wherein the feeder cells comprise fibroblast cells. 
   
   
       59 . The method of  claim 58 , wherein said fibroblast cells are selected from human embryonic fibroblast cells (HEF), umbilical cord derived fibroblast cells and foreskin derived fibroblast cells. 
   
   
       60 . A culture system for maintenance of a SC in an undifferentiated state, the culture system comprising Neurobasal™ medium. 
   
   
       61 . The culture system of  claim 60 , comprising N2 supplement or a modification of N2 supplement. 
   
   
       62 . The culture system of  claim 60 , for propagation of SC in a suspension or in flat colonies. 
   
   
       63 . The culture system of  claim 60 , wherein said SC are human SC. 
   
   
       64 . The culture system of  claim 60 , wherein said SC are embryonic SC. 
   
   
       65 . The culture system of  claim 60 , comprising one or more growth factors for promoting growth of said undifferentiated hESC in culture. 
   
   
       66 . The culture system of  claim 60 , comprising an extracellular matrix (ECM). 
   
   
       67 . A cell culture of undifferentiated human embryonic SC obtained by incubating a cluster of cells from inside a blastocyst with a culture system according to  claim 60 . 
   
   
       68 . The cell culture of  claim 67 , wherein said cells are maintained in a suspension or as flat colonies. 
   
   
       69 . A method for maintaining SC in an undifferentiated state, the method comprising incubating said cells with a culture system comprising Neurobasal™ medium. 
   
   
       70 . The method of  claim 69 , comprising supplementing said medium with N2 supplement or a modification of N2 supplement. 
   
   
       71 . The method of  claim 69 , wherein said cells propagate in a suspension or as flat colonies. 
   
   
       72 . A method of obtaining a cell culture comprising cells obtained from human umbilical cord tissue, the human umbilical cord derived cells being capable of maintaining stem cells (SC) in an undifferentiated state when co-cultured therewith, the method comprises: (a) isolating umbilical cord cells from umbilical cord tissue; (b) culturing said umbilical cord cells in a culture medium including serum. 
   
   
       73 . The method of  claim 72 , wherein said umbilical cord tissue is obtained from healthy pregnant women undergoing elective Cesarean sections at term. 
   
   
       74 . The method of  claim 72 , wherein said umbilical cord cells are obtainable by mincing said umbilical cord tissue into small pieces. 
   
   
       75 . The method of  claim 74 , wherein the minced umbilical cord tissue pieces are fixed to walls of a container in the presence of culture medium, and allowed to incubate undisturbed for a number of weeks until the fibroblast cells begin to migrate out of the umbilical cord pieces. 
   
   
       76 . The method of  claim 72 , wherein said serum is human serum. 
   
   
       77 . The method of  claim 72 , wherein said serum and/or said serum replacement are provided at a concentration of at least 10%. 
   
   
       78 . The method of  claim 77 , wherein said serum and/or said serum replacement are provided at a concentration of 20%.