Method of inducing beta cells from urine-derived cells using small molecules

1. A method of producing induced beta cells from urine-derived cells, the method comprising:

(a) providing urine-derived cells; wherein the urine-derived cells express human nuclear antigen, CD13, vimentin and E-cadherin

(b) inducing the urine-derived cells provided in step (a) to obtain induced endoderm cells by an induction process consisting of culturing said urine-derived cells in a primary induction culture medium comprising an effective amount of a combination of small molecule reprogramming factor(s) comprising a definitive endoderm inducer, a glycogen synthase kinase 3 (GSK3) inhibitor and Vitamin C for a first period of time;

(c) inducing the induced endoderm cells obtained in step (b) by culturing said induced endoderm cells in a secondary induction culture medium comprising an effective amount of at least one small molecule reprogramming factor(s) selected from the group consisting of a PKC activator, a sonic hedgehog (SSH) inhibitor, a retinoic acid receptor (RAR) agonist, Vitamin C, an activin receptor-like kinase (ALK) receptor inhibitor, a Pdx1 inducer and a bone morphogenetic protein (BMP) inhibitor for a second period of time to obtain induced pancreatic precursor cells; and

(d) inducing the induced pancreatic precursor cells obtained in step (c) by culturing said pancreatic precursor cells in a tertiary induction culture medium comprising an effective amount of at least one small molecule reprogramming factor(s) selected from the group consisting of a mitogen-activated protein kinase kinase (MEK) inhibitor, Vitamin C, a notch inhibitor, an ALK receptor inhibitor, triiodothyronine, an RAR agonist, and a SSH inhibitor for a third period of time to obtain induced beta cells; and

wherein the method excludes the use of reprogramming factors that are not small molecules.

2. The method of claim 1 , wherein the second period of time consists of a first portion and a second portion, and wherein the secondary induction medium comprises an effective amount of at least one small molecule reprogramming factor(s) selected from the group consisting of:

a PKC activator, a sonic hedgehog (SSH) inhibitor, a retinoic acid receptor (RAR) agonist, Vitamin C, an activin receptor-like kinase (ALK) receptor inhibitor, and a Pdx1 inducer for a first portion of the second period of time; and

a PKC activator, a sonic hedgehog (SSH) inhibitor, Vitamin C, an activin receptor-like kinase (ALK) receptor inhibitor, and a Pdx1 inducer for a second portion of the second period of time.

3. The method of claim 1 , wherein the second period of time consists of a first portion, a second portion and a third portion, and wherein the secondary induction medium comprises an effective amount of at least one small molecule reprogramming factor(s) selected from the group consisting of:

Vitamin C for the first portion of the second period of time;

Vitamin C, a RAR agonist, a SSH inhibitor, a PKC activator and a BMP inhibitor for a second portion of the second period of time; and

Vitamin C, a RAR agonist, and a SSH inhibitor for a third portion of the second period of time.

4. The method of claim 1 , wherein the tertiary induction medium comprises an effective amount of a combination of small molecule reprogramming factor(s) comprising the MEK inhibitor, Vitamin C, and the notch inhibitor.

5. The method of claim 1 , wherein the third period of time consists of a first portion, a second portion and a third portion, and wherein the tertiary induction medium comprises an effective amount of at least one small molecule reprogramming factor(s) selected from the group consisting of:

Vitamin C, a RAR agonist, a SSH inhibitor, a notch inhibitor, an ALK receptor inhibitor, and triiodothyronine for the first portion of the third period of time,

Vitamin C, a RAR agonist, a notch inhibitor, an ALK receptor inhibitor, and triiodothyronine for the second portion of the third period of time, and

Vitamin C and triiodothyronine for the third portion of the third period of time.

6. The method of claim 1 , wherein:

the primary induction medium comprises an effective amount of IDE1, lithium chloride and Vitamin C;

the secondary induction medium comprises an effective amount of

Indolactam V, cyclopamine-KAAD, Vitamin C, retinoic acid, A83-01 and BRD 7552 for a first portion of the second period of time, and

Indolactam V, cyclopamine-KAAD, Vitamin C, A83-01 and BRD 7552 for a second portion of the second period of time; and

the tertiary induction medium comprises an effective amount of SB203580, Vitamin C and DAPT.

7. The method of claim 1 , wherein:

the primary induction medium comprises an effective amount of IDE1, lithium chloride and Vitamin C;

the secondary induction medium comprises an effective amount of

Vitamin C for a first portion of the second period of time;

Vitamin C, RA, cyclopamine-KAAD, Indolactam V and dorsomorphin for a second portion of the second period of time; and

Vitamin C, RA, and cyclopamine-KAAD for a third portion of the second period of time; and

the tertiary induction medium comprises an effective amount of

Vitamin C, RA, cyclopamine-KAAD, DAPT, A83-01, and triiodothyronine for a first portion of the third period of time,

Vitamin C, RA, DAPT, A83-01, and triiodothyronine for a second portion of the third period of time, and

Vitamin C and triiodothyronine for a third portion of the third period of time.

8. A method of producing induced endoderm cells from urine-derived cells, the method comprising:

(a) providing urine-derived cells; wherein the urine-derived cells express human nuclear antigen, CD13, vimentin and E-cadherin and

(b) inducing the urine-derived cells provided in step (a) to obtain induced endoderm cells by an induction process consisting of culturing said urine-derived cells in a primary induction culture medium comprising an effective amount of a combination of small molecule reprogramming factor(s) comprising a definitive endoderm inducer, a glycogen synthase kinase 3 (GSK3) inhibitor and Vitamin C for a first period of time; and

wherein the method excludes the use of reprogramming factors that are not small molecules.

9. A method of producing induced pancreatic precursor cells from urine-derived cells, the method comprising:

(a) providing urine-derived cells; wherein the urine-derived cells express human nuclear antigen, CD13, vimentin and E-cadherin

(b) inducing the urine-derived cells provided in step (a) to obtain induced endoderm cells by an induction process consisting of culturing said urine-derived cells in a primary induction culture medium comprising an effective amount of a combination of small molecule reprogramming factor(s) comprising a definitive endoderm inducer, a glycogen synthase kinase 3 (GSK3) inhibitor and Vitamin C for a first period of time to obtain induced endoderm cells; and

(c) inducing the induced endoderm cells obtained in step (b) by culturing said induced endoderm cells in a secondary induction culture medium comprising an effective amount of at least one small molecule reprogramming factor(s) selected from the group consisting of a PKC activator, a sonic hedgehog (SSH) inhibitor, a retinoic acid receptor (RAR) agonist, Vitamin C, an activin receptor-like kinase (ALK) receptor inhibitor, a Pdx1 inducer and a bone morphogenetic protein (BMP) inhibitor for a second period of time to obtain induced pancreatic precursor cells; and

wherein the method excludes the use of reprogramming factors that are not small molecules.

10. The method of claim 1 , wherein the urine-derived cells are obtained from a urine sample of a subject.

11. The method of claim 1 , wherein step (a) further comprises expanding the urine-derived cells by culturing the urine-derived cells in an appropriate tissue culture medium.