SELECTIVE CELL THERAPY FOR THE TREATMENT OF RENAL FAILURE

• US 20100104544A1
• Filed: 11/13/2009
• Published: 04/29/2010
• Est. Priority Date: 06/08/2007
• Status: Active Grant

• Current Assignee: Wake Forest University Health Sciences
• Original Assignee: Wake Forest University Health Sciences
• Inventors: Atala, Anthony, Yoo, James J.
• Granted Patent: US 10,590,391 B2
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/22 Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 5/0686 Kidney cells

Abstract

Provided herein are isolated populations of kidney cells harvested from differentiated cells of the kidney, wherein the cells have been expanded in vitro. The kidney cells preferably produce erythropoietin (EPO). The kidney cells may also be selected based upon EPO production. Methods of producing an isolated population of EPO producing cells are also provided, and methods of treating a kidney disease resulting in decreased EPO production in a patient in need thereof are provided, including administering the population to the patient, whereby the cells express EPO in vivo in an oxygen tension-dependent manner.

Claims

1. An isolated population of cells comprising differentiated kidney cells passaged in vitro, wherein said population has oxygen tension-dependent erythropoietin (EPO) expression.

2. The population of cells according to claim 1, wherein said cells have an increase in EPO expression as measured by an increase in EPO mRNA when under hypoxic conditions as compared to normoxic conditions, and have a decrease in EPO expression as measured by a decrease in EPO mRNA expression when cells return to normoxic conditions from hypoxic conditions.

3. The population of cells according to claim 1, wherein said cells have an increase in EPO expression as measured by a 2- to 20-fold increase in EPO mRNA after incubation of said cells at 70% confluencey for 24 hours:

under hypoxic conditions of;

a humidified incubator at 37°

C., at atmospheric pressure, and with 1% O₂, 5% CO₂ and 94% N₂, as compared to incubation under normoxic conditions of;

a humidified incubator at 37°

C., at atmospheric pressure, and with 5% CO₂ and 95% atmospheric air,

wherein said incubation media is a 1;

1 mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbeeco'"'"'s Modified Eagle'"'"'s Medium (DMEM) based media, wherein said DMEM based media is ¾

DMEM and ¼

HAM'"'"'s F12 nutrient mixture supplemented with 5% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 1% glutamine 100×

, 1 ml of 0.4 μ

g/ml hydrocortisone, 0.5 ml of a 10<sup>−

10</sup> M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μ

g/ml epidermal growth factor (EGF) solution,

and wherein said cells have been washed with said incubation media and fresh incubation media applied within one hour prior to said incubation for 24 hours.

4. The population of cells according to claim 1, wherein said cells have a decrease in EPO expression as measured by a 2- to 20-fold decrease in EPO mRNA after incubation of said cells at 70% confluency for 20 hours:

under hypoxic conditions for 10 hours, said hypoxic conditions being;

a humidified incubator at 37°

C., at atmospheric pressure, and with 1% O₂, 5% CO₂ and 94% N₂,

followed by incubation for 10 hours under normoxic conditions of;

a humidified incubator at 37°

C., at atmospheric pressure, and with 5% CO₂ and 95% atmospheric air;

as compared to incubation for 20 hours under said hypoxic conditions given above,

wherein said incubation media is a 1;

1 mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbecco'"'"'s Modified Eagle'"'"'s Medium (DMEM) based media, wherein said DMEM based media is ¾

DMEM and ¼

HAM'"'"'s F12 nutrient mixture supplemented with 5% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 1% glutamine 100×

, 1 ml of 0.4 μ

g/ml hydrocortisone, 0.5 ml of a 10<sup>−

10</sup> M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μ

g/ml epidermal growth factor (EGF) solution,

and wherein said cells have been washed with said incubation media and fresh incubation media applied within one hour prior to said incubation for 20 hours.

5. The population of cells according to claim 1, wherein said population has been passaged from 1 to 20 times.

6. The population of cells according to claim 1, wherein said population has been passaged from 1 to 10 times.

7. The population of cells according to claim 1, wherein said population has been passaged from 1 to 5 times.

8. The population of cells according to claim 1, wherein said population has been selected for EPO production.

9. The population of cells according to claim 1, subject to the proviso that said cells are not transfected with an exogenous DNA encoding a polypeptide.

10. A composition comprising the population of cells according to claim 1 and a pharmaceutically acceptable carrier.

25. A method of treating anemia in a patient in need thereof, said method comprising:

providing a composition comprising the population of cells according to claim 10; and

administering said composition to said patient in an amount effective to treat said anemia.

26. The method of claim 25, wherein said administering step is carried out by injecting or implanting said composition into said patient.

27. The method of claim 25, wherein said anemia is selected from the group consisting of:

an anemia of renal failure, an anemia of end-stage renal disease, an anemia of a chemotherapy, an anemia of a radiation therapy, an anemia of chronic infection, an anemia of an autoimmune disease, an anemia of rheumatoid arthritis, an anemia of AIDS, an anemia of a malignancy, an anemia of prematurity, an anemia of hypothyroidism, an anemia of malnutrition, and an anemia of a blood disorder.

11. A method of treating a kidney disease resulting in decreased erythropoietin (EPO) production in a patient in need thereof, said method comprising:

providing a composition comprising differentiated kidney cells and a pharmaceutically acceptable carrier; and

administering said composition to said patient,

wherein said cells have oxygen tension-dependent EPO expression.

12. The method of claim 11, wherein said pharmaceutically acceptable carrier comprises a collagen gel.

13. The method of claim 11, wherein said cells have been passaged from 1 to 20 times prior to said administering.

14. The method of claim 11, wherein said cells have been passaged from 1 to 10 times prior to said administering.

15. The method of claim 11, wherein said cells have been passaged from 1 to 5 times prior to said administering.

16. The method of claim 11, wherein said cells have been selected for EPO production prior to said administering.

17. The method of claim 11, subject to the proviso that said cells are not transfected with an exogenous DNA encoding a polypeptide.

18. The method of claim 11, wherein said administering is carried but by injecting said composition into the kidney or liver of said patient.

19. The method of claim 11, wherein said administering step is carried out by injecting or infusing said composition intravascularly.

20. The method of claim 11, where said administering step is carried out by infusing said composition into a portal vein of said patient.

21. The method of claim 11, wherein said pharmaceutically acceptable carrier comprises a biodegradable scaffold administering step is carried out by implanting said composition into the kidney of said patient.

22. The method of claim 11, wherein said kidney disease is an anemia selected from the group consisting of:

an anemia of renal failure, an anemia of end-stage renal disease, an anemia of a chemotherapy, an anemia of a radiation therapy, an anemia of chronic infection, an anemia of an autoimmune disease, an anemia of rheumatoid arthritis, an anemia of AIDS, an anemia of a malignancy, an anemia of prematurity, an anemia of hypothyroidism, an anemia of malnutrition, and an anemia of a blood disorder.

23. The method of claim 11, wherein said EPO producing cells are mammalian.

24. The method of claim 11, wherein said EPO producing cells are human.

28. A method of selecting cells for erythropoietin (EPO) production comprising:

providing a composition comprising differentiated kidney cells; and

selecting EPO producing cells from said composition, wherein said EPO producing cells have oxygen tension-dependent EPO expression.

29. The method of claim 28, wherein said EPO producing cells have an increase in EPO expression as measured by an increase in EPO mRNA when under hypoxic conditions as compared to normoxic conditions, and have a decrease in EPO expression as measured by a decrease in EPO mRNA when cells return to normoxic conditions from hypoxic conditions.

30. The method of claim 28, wherein said EPO producing cells have an increase in EPO expression as measured by a 2- to 20-fold increase in EPO mRNA after incubation of said cells at 70% confluencey for 24 hours:

under hypoxic conditions of;

a humidified incubator at 37°

C., at atmospheric pressure, and with 1% O₂, 5% CO₂ and 94% N₂,

as compared to incubation under normoxic conditions of;

a humidified incubator at 37°

C., at atmospheric pressure, and with 5% CO₂ and 95% atmospheric air,

wherein said incubation media is a 1;

1 mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbecco'"'"'s Modified Eagle'"'"'s Medium (DMEM) based media, wherein said DMEM based media is ¾

DMEM and ¼

HAM'"'"'s F12 nutrient mixture supplemented with 5% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 1% glutamine 100×

, 1 ml of 0.4 μ

g/ml hydrocortisone, 0.5 ml of a 10<sup>−

10</sup> M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μ

g/ml epidermal growth factor (EGF) solution,

and wherein said cells have been washed with said incubation media and fresh incubation media applied within one hour prior to said incubation for 24 hours.

31. The method of claim 28, wherein said EPO producing cells have a decrease in EPO expression as measured by a 2- to 20-fold decrease in EPO mRNA after incubation of said cells at 70% confluency for 20 hours:

under hypoxic conditions for 10 hours, said hypoxic conditions being;

a humidified incubator at 37°

C., at atmospheric pressure, and with 1% O₂, 5% CO₂ and 94% N₂,

followed by incubation for 10 hours under normoxic conditions of;

a humidified incubator at 37°

C., at atmospheric pressure, and with 5% CO₂ and 95% atmospheric air;

as compared to incubation for 20 hours under said hypoxic conditions given above,

wherein said incubation media is a 1;

1 mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbecco'"'"'s Modified Eagle'"'"'s Medium (DMEM) based media, wherein said DMEM based media is ¾

DMEM and ¼

HAM'"'"'s F12 nutrient mixture supplemented with 5% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 1% glutamine 100×

, 1 ml of 0.4 μ

g/ml hydrocortisone, 0.5 ml of a 10<sup>−

10</sup> M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μ

g/ml epidermal growth factor (EGF) solution,

and wherein said cells have been washed with said incubation media and fresh incubation media applied within one hour prior to said incubation for 20 hours.

32. A kit comprising:

a media for growing differentiated kidney cells;

an antibody or active fragment thereof which selectively binds EPO; and

optionally, instructions for use.

33. The kit of claim 32, wherein said media comprises a 1:

1 mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbecco'"'"'s Modified Eagle'"'"'s Medium (DMEM) based media, wherein said DMEM based media is % DMEM and ¼

HAM'"'"'s F12 nutrient mixture supplemented with 5% serum, 1% antibiotic, 1% glutamine 100×

, 1 ml of 0.4 μ

g/ml hydrocortisone, 0.5 ml of a 10<sup>−

10</sup> M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μ

g/ml epidermal growth factor (EGF) solution.