A method for the regeneration and differentiation of human perinephric fat derived mesenchymal stromal cells into astroglial, renal, neuronal and pancreatic progenitor cells

A method for differentiating the isolated human perinephric fat derived stem/stromal cells (hPNF-MSCs) into neural cells, the method comprises the steps of:

• generating spheres with a basic fibroblast growth factor (bFGF) at a concentration of 1 ng/ml in a DMEM-MO medium, and wherein the DMEM-KO medium comprises a fetal bovine serum (FBS) at a concentration of 1% v/v;
• incubating the generated spheres in the medium for 24 hours;
• replacing the growth medium with a neural induction medium, and wherein the neural induction medium comprises a DMEM-FI 2 culture medium, and wherein the DMEM-F12 medium comprises fetal bovine serum (FBS) at a concentration of 1% v/v, a N2 at a concentration of 1% v/v, a B27 at a concentration of 2% and a bFGF at a concentration of 20 ng/mL, and wherein the FBS is a nutritional supplement, and wherein the N2 is a cocktail of the growth factor, and wherein the N2 comprises an insulin at a concentration of 5 μg/ml, a transferrin at a concentration of 100 μg/ml, a progesterone at a concentration of 20 nM, a putrescine at a concentration of 100 μM, a selenium at a concentration of 30 nM, a B27 at a concentration of 2% v/v, and bFGF at a concentration of 20 ng/mL, and wherein the selenium is added for growth and maintenance of neurons in vitro, and wherein the B27 is a growth supplement for neurons in vitro, and wherein the bFGF is a cell differentiating agent, and wherein the bFGF is a cell differentiating agent, and wherein the bFGF induces cell differentiation of the hPNF-MSCs into neuronal progenitor cells, and wherein the bFGF targets Wnt pathway;
• replenishing the neural induction medium after every 3-4 days;
• fixing the cells with at a concentration of 4% v/v for characterization;
• and
• performing induction procedure on a monolayer culture of the cells.

A method for differentiating the isolated human perinephric fat derived stem/stromal cells (hPNF-MSCs) into astroglial cells, the method comprises the steps of:

• generating spheres with a basic fibroblast growth factor (bFGF) at a concentration of 1 nm/mL in a DMEM-KO medium, and wherein the DMEM-KO medium is supplemented with a fetal bovine serum (FBS) at a concentration of 1% v/v;
• incubating the generated spheres in the medium for 24 hours;
• replacing the growth medium with an astroglial induction medium and a L-thyroxine, and wherein the astroglial induction medium comprises a FBS at a concentration of 1% v/v, a N2 at a concentration of 1% v/v and a T4 at a concentration of 30 ng/mL, and wherein the FBS is a nutritional supplement, and wherein the N2 is a cocktail of the growth factor, and wherein the N2 comprises an insulin at a concentration of 5 μg/ml, a progesterone at a concentration of 20 nM, a putrescine at a concentration of 100 μM, a selenium at a concentration of 30 nM, a B27 at a concentration of 2% v/v and a bFGF at a concentration of 20 ng/mL, and wherein the selenium is added for growth and maintenance of astroglial cells in vitro, and wherein the B27 is a growth supplement for astroglial cells in vitro, and wherein the bFGF is a cell differentiating agent, and wherein the bFGF induces cell differentiation of the hPNF-MSCs into astroglial progenitor cells, and wherein the T4 induces the differentiation of hPNF-MSCs into astroglial cells, and wherein the bFGF targets Wnt pathway;
• replenishing the astroglial induction medium after every 3-4 days;
• fixing the cells with a 4% PFA for characterization;
• and
• performing induction procedure on a monolayer culture of the cells.

A method for differentiating isolated human perinephric fat derived stem/stromal cells (hPNF-MSCs) into renal cells, the method comprises the steps of:

• culturing the hPNF-MSC on autoclaved coverslips in 35 mm culture dishes comprising a renal epithelial growth medium;
• incubating the culture dishes at 37° C. in a CO2 incubator for 17 days for identifying the differentiation of hPNF-MSC to metanephric mesenchyme, and wherein the CO2 is at a concentration of 5%;
• incubating the culture dishes at 37° C. in a CO2 incubator for 22 days for identifying the differentiation of hPNF-MSC to podocyte and proximal convulated tubule, and wherein the CO2 is at a concentration of 5%;
• replenishing the culture medium after every 48 hours;
• wherein the renal epithelium growth medium comprises a BMP2 at a concentration of 10 nm/ml, a retinoic acid at a concentration of 0.1 μM/ml and an Activin A at a concentration of 10 ng/ml, and wherein the BMP2, the retinoic acid, and the Activin A are the growth factors which induce differentiation in the hPNF-MSC to form renal cells, and wherein the Activin A induces a mesoderm specification, and wherein the retinoic acid induces the mesoderm patterning, and wherein the retinoic acid targets retinoid receptors RAR, and wherein the BMP2 induces nephrogenesis, and wherein the BMP2 is expressed in a metanephric mesenchyme, and wherein the BMP2 induces epithelialization of a metanephric mesenchyme;
• performing syringe filtration of the renal epithelium differentiation medium using a 0.22 micron filter;
• incubating the hPNF-MSCs at a temperature of 37° C. in a CO2 incubator for 17 days to identify the differentiation of hPNF-MSCs into metanephric mesenchyme cells, and wherein the concentration of CO2 is 5%;
• incubating the hPNF-MSCs at a temperature of 37° C. in an incubator for 22 days for a podocyte and a proximal convulated tubule differentiation, wherein the incubator has CO2 at a concentration of 5%;
• and
• replenishing the renal epithelium differentiation medium after every 48 hours.

A method for differentiating isolated human perinephric fat derived stem/stromal cells (hPNF-MSCs) into islet cells, the method comprises the steps of:

• seeding the human perinephric mesenchymal stem cells (hPNF-MSCs) from a passage 3 (P3) in three non-adherent cell culture dishes/plates;
• labeling two cell culture dishes/plates as cell differentiation culture dish/plate, and wherein the hPNF-MSCs are differentiated into pancreatic progenitor cell clusters in the cell differentiation culture dish/plates;
• labeling one cell culture dish/plate as control dish/plate, and wherein the control dish/plate is an un-induced culture dish/plate;
• supplementing the control cell culture dish/plate (un-induced culture dish/plate) with a cell culture medium, and wherein the cell culture medium comprises Dulbecco's Modified Eagle's Medium-Knock Out (DMEM-KO);
• supplementing the two cell differentiation plates/dishes with a culture medium and wherein the cell culture medium comprises Dulbecco's Modified Eagle's Medium-Knock Out (DMEM-KO), a bovine serum albumin (BSA) in a concentration of 1.5% v/v, insulin-transferrin-selenium (ITS) in a concentration of 1×,
• incubating the two cell differentiation culture dish/plate and one control cell culture dish/plate at 37° C. for four days in a CO2 incubator, and wherein the CO2 incubator is filled with CO2 at a concentration of 5%;
• collecting a plurality of floating cells in a culture medium in a centrifuge tube after four days;
• allowing the cells to settle down in the centrifuge tube for 15-20 minutes, and wherein cells settle down to form a pellet and the culture medium forms a supernatant and wherein the cell pellet is collected;
• decanting the supernatant;
• re-seeding the cells from the cell pellet in a day 4 medium and wherein the 4-days old medium comprises a Dulbecco's Modified Eagle's Medium (DMEM-KO), bovine serum albumin (BSA) in a concentration of 1.5%, insulin-transferrin-selenium (ITS) in a concentration of 1×, a bovine serum albumin (BSA) and Taurine at a concentration of 0.3 mM.
• incubating the cells at preset temperature in the CO2 incubator for further three days, and wherein the preset temperature is 37° C. and wherein the cells are incubated for a total period of seven days, and wherein the CO2 incubator is filled with CO2 at a concentration of 5%;
• feeding the cells with a fresh day 4 medium on day 7;
• incubating the cells at preset temperature in a CO2 incubator for further three days, and wherein the preset temperature is 37° C. and wherein the cells are incubated for a total period often days, and wherein the CO2 incubator is filled with CO2 at a concentration of 5%;
• collecting floating cell clusters with culture medium in a sterile centrifuge tube after 10 days;
• allowing the cells to settle for 15-20 minutes at a room temperature, and wherein the cells settle down to form the pellet, and wherein the culture medium forms the supernatant;
• decanting the supernatant;
• wherein the cell pellet comprises cell clusters, and wherein the cell clusters are re-seeded in a medium comprising a DMEM-KO, BSA at a concentration of 1.5% v/v, ITS at a concentration of 1×, 100 nM nicotine amide at a concentration of 100 nM, taurine at a concentration of 3 nM and a glucagon like peptide 1 (GLP 1) at a concentration of 100 nM;
• subjecting the cells for a dithiozone (DTZ) staining analysis and an immunofluorescence analysis;
• and
• wherein the cell clusters are islet cell clusters.