Reprogrammed cells and methods of production and use thereof

Patent metadata

Field	Value
Publication number	US-2016115455-A1
Application number	US-201414891897-A
Country	US
Kind code	A1
Filing date	May 16, 2014
Priority date	May 17, 2013
Publication date	Apr 28, 2016
Grant date	—

Abstract

The present invention relates generally to a secondary reprogramming technique and its uses. More particularly, it concerns immortalized secondary somatic cells, secondary induced pluripotent stem cells (iPSCs), secondary induced multipotent progenitor cells (iMPCs) and tertiary somatic cells derived therefrom, and methods of using these cells to assess the effects of agents on cells and for medical treatment of subjects.

First claim

1 . A cell that i) contains an inducible first polycistronic exogenous nucleic acid encoding one or more reprogramming factors operably linked to a first regulatory sequence; and ii) expresses a second exogenous nucleic acid encoding an immortalizing factor operably linked to a second regulatory sequence; wherein the cell is capable of proliferating through at least 10, 20, 25, 30, 35, or even 40 or more population doublings.

2 . The cell of claim 1 , wherein the cell is a human cell or a mouse cell.

3 . The cell of claim 1 , wherein the cell is a somatic cell, a fibroblast, a keratinocyte or an adult stem cell.

4 - 6 . (canceled)

7 . The cell of claim 1 , wherein the one or more reprogramming factors are pluripotency factors selected from Oct4, KLF4, Myc, and Sox2, and combinations thereof.

8 . The cell of claim 1 , wherein the immortalizing factor is hTERT.

9 . The cell of claim 1 , wherein the cell does not express the reprogramming factors encoded in the inducible first polycistronic exogenous nucleic acid.

10 . The cell of claim 1 , wherein the second exogenous nucleic acid is capable of being deactivated when the first polycistronic exogenous nucleic acid is expressed, or wherein the second exogenous nucleic acid is inducible.

11 . (canceled)

12 . A cell produced by inducing expression of the reprogramming factors encoded in the inducible first polycistronic exogenous nucleic acid in a cell of claim 1 .

13 . A population of substantially uniform cells that i) express an inducible first polycistronic exogenous nucleic acid encoding one or more reprogramming factors operably linked to a first regulatory sequence; and ii) express a second exogenous nucleic acid encoding an immortalizing factor operably linked to a second regulatory sequence; wherein the population optionally comprises at least 4 million, 8 million, 16 million, 32 million, 64 million, or even 128 million or more substantially uniform cells.

14 . (canceled)

15 . A method of reprogramming a cell, comprising inducing expression of the reprogramming factors encoded in the inducible first polycistronic exogenous nucleic acid in a cell of claim 1 ; wherein the method is optionally carried out on a population of at least 4 million, 8 million, 16 million, 32 million, 64 million, or even 128 million or more substantially uniform cells.

16 . (canceled)

17 . A method of producing an immortalized secondary cell, comprising:
i) introducing into an initial cell an inducible first polycistronic exogenous nucleic acid encoding pluripotency factors operably linked to a first regulatory sequence; ii) inducing expression of the pluripotency factors encoded in the inducible first polycistronic exogenous nucleic acid to produce a de-differentiated cell; iii) exposing the de-differentiated cell to differentiation agents to produce a secondary cell; and iv) introducing into the secondary cell a second exogenous nucleic acid encoding an immortalizing factor operably linked to a second regulatory sequence; and v) causing the secondary cell to express the immortalizing factor; wherein the method may optionally further comprise proliferating the immortalized secondary cells for at least 10, 15, 20, 25, 30, 35, or even 40 or more population doublings.

18 . (canceled)

19 . A method of claim 17 , further comprising inducing expression of the pluripotency factors encoded in the inducible first polycistronic exogenous nucleic acid in all or a portion of the proliferated secondary cells.

20 . (canceled)

21 . The method of claim 19 , further comprising inducing expression of the pluripotency factors encoded in the inducible first polycistronic exogenous nucleic acid in the secondary cell that expresses or expressed the immortalizing factor, wherein inducing the expression of the pluripotency factors causes the immortalizing factor to stop being expressed.

22 - 25 . (canceled)

26 . The method of claim 17 , wherein the initial cell is a somatic cell, a fibroblast, or a keratinocyte.

27 . (canceled)

28 . The method of claim 26 , wherein the somatic cell is an adult stem cell, a hematopoietic stem cell, neural stem cell, or mesenchymal stem cell.

29 - 32 . (canceled)

33 . A cell produced by the method of claim 15 .

34 . A method of producing an engineered cell, comprising:
i) introducing into an initial cell an inducible exogenous nucleic acid encoding one or more reprogramming factors operably linked to a first regulatory sequence and an exogenous nucleic acid encoding an immortalizing factor operably linked to a second regulatory sequence; ii) inducing expression of the one or more reprogramming factors encoded in the inducible exogenous nucleic acid; and iii) causing the cell to express the immortalizing factor; and wherein the method optionally further comprises proliferating the cell expressing the immortalizing factor for at least 10, 15, 20, 25, 30, 35, or even 40 or more population doublings.

35 - 38 . (canceled)

39 . A method of producing an immortalized secondary cell, comprising:
i) introducing into an initial cell an inducible exogenous nucleic acid encoding one or more reprogramming factors operably linked to a first regulatory sequence; ii) inducing expression of the reprogramming factors encoded in the inducible exogenous nucleic acid to produce a reprogrammed cell; iv) introducing into the reprogrammed cell an exogenous nucleic acid encoding an immortalizing factor operably linked to a second regulatory sequence, and v) causing the reprogrammed cell to express the immortalizing factor; and wherein the method optionally further comprises proliferating the cell expressing the immortalizing factor for at least 10, 15, 20, 25, 30, 35, or even 40 or more population doublings.

40 . A method of producing an immortalized secondary cell, comprising:
i) introducing into an initial cell an exogenous nucleic acid encoding an immortalizing factor operably linked to a first regulatory sequence; ii) causing the cell to express the immortalizing factor, thereby producing an immortalized cell; iii) introducing into the immortalized cell an inducible exogenous nucleic acid encoding one or more reprogramming factors operably linked to a second regulatory sequence, and iv) inducing expression of the one or more reprogramming factors encoded in the inducible exogenous nucleic acid to produce a reprogrammed cell; and wherein the method optionally further comprises proliferating the cell expressing the immortalizing factor for at least 10, 15, 20, 25, 30, 35, or even 40 or more population doublings.

41 . (canceled)

42 . A method of claim 34 , further comprising inducing expression of the one or more reprogramming factors in the secondary cell that expresses the immortalizing factor or has been induced to express the immortalizing factor, wherein inducing the expression of the one or more reprogramming factors causes the immortalizing factor to stop being expressed.

43 - 46 . (canceled)

47 . The method of claim 34 , wherein the immortalizing factor is constitutively expressed after being introduced into the cell.

48 - 55 . (canceled)

56 . A cell produced by the method of claim 34 .

57 . A method of identifying

Assignees

• Broad Inst Inc
• Harvard College

Inventors

• Mikkelsen Tarjei
• Cacchiarelli Davide

Classifications

• C12N2501/602

  Sox-2 · CPC title

• C12N2501/606

  c-Myc · CPC title

• G01N33/5073

  Stem cells · CPC title

• C12N2501/603

  Oct-3/4 · CPC title

• C12N5/0696 Primary

  Artificially induced pluripotent stem cells, e.g. iPS · CPC title