Methods and compositions for producing oogonium-like cells
Patent Information
- Application Number
- RU2024129296
- Authority / Receiving Office
- RU · RU
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2023-03-30
- Publication Date
- 2026-09-09
Claims
1. A pluripotent stem cell (PSC) comprising: an engineered polynucleotide comprising an open reading frame encoding a protein selected from zinc finger protein 281 (ZNF281), LIM homeobox 8 (LHX8), and spermatogenesis- and oogenesis-specific helix-loop-helix-containing protein 1 (SOHLH1).
2. The PSC of claim 1, wherein the PSC comprises a designed polynucleotide comprising an open reading frame encoding ZNF281.
3. The PSC of claim 1, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding LHX8.
4. The PSC of claim 1, wherein the PSC comprises a designed polynucleotide comprising an open reading frame encoding SOHLH1.
5. The PSC of claim 1, wherein the PSC expresses or overexpresses: ZNF281; LHX8; SOHLH1; ZNF281 and LHX8; ZNF281 and SOHLH1; LHX8 and SOHLH1; or ZNF281, LHX8 and SOHLH1.
6. The PSC of claim 1, wherein the PSC further comprises an engineered polynucleotide comprising an open reading frame encoding a folliculogenesis-specific transcription factor BHLH (FIGLA) protein, wherein optionally the PSC expresses or overexpresses FIGLA.
7. The PSC of claim 6, wherein the PSC further comprises: an engineered polynucleotide comprising an open reading frame encoding distal-less homeobox protein 5 (DLX5); and an engineered polynucleotide comprising an open reading frame encoding a hematopoietically expressed homeobox protein (HHEX), wherein optionally the PSC expresses or overexpresses DLX5 protein and HHEX protein.
8. The PSC of claim 7, wherein the PSC further comprises: an engineered polynucleotide comprising an open reading frame encoding the DDX4 protein; an engineered polynucleotide comprising an open reading frame encoding the DAZL protein; and an engineered polynucleotide comprising an open reading frame encoding the BOLL protein, wherein optionally the PSC expresses or overexpresses the DDX4 protein, the DAZL protein, and the BOLL protein.
9. The PSC of claim 1, wherein the open reading frame of the constructed polynucleotide is operably linked to a heterologous promoter.
10. The PSC of claim 9, wherein the heterologous promoter is an inducible promoter.
11. A pluripotent stem cell (PSC) comprising: a protein selected from ZNF281, LHX8 and SOHLH1, wherein the protein is overexpressed.
12. The PSC of claim 11, wherein the PSC expresses or overexpresses: ZNF281; LHX8; SOHLH1; ZNF281 and LHX8; ZNF281 and SOHLH1; LHX8 and SOHLH1; or ZNF281, LHX8 and SOHLH1.
13. The PSC of claim 12, wherein the PSC further comprises a FIGLA protein, wherein optionally the PSC expresses or overexpresses FIGLA.
14. The PSC of claim 13, wherein the PSC further comprises: a DLX5 protein and an HHEX protein, wherein optionally the PSC expresses or overexpresses the DLX5 protein and the HHEX protein.
15. The PSC of claim 14, wherein the PSC further comprises: a DDX4 protein, a DAZL protein and a BOLL protein, wherein optionally the PSC expresses or overexpresses the DDX4 protein, the DAZL protein and the BOLL protein.
16. The PSC of claim 1, wherein the PSC is a human PSC.
17. The PSC of claim 1, wherein the PSC is an induced PSC (iPSC).
18. The PSC of claim 1, wherein the PSC comprises 1-20, optionally 8-10 copies of a designed polynucleotide comprising an open reading frame encoding a protein selected from ZNF2 81, LHX8 and SOHLH1.
19. A composition comprising a PSC population according to claim 1.
20. The composition of claim 19, wherein the population comprises at least 2500 PSC / cm 2 .
21. A method comprising: culturing in a culture medium a population of pluripotent stem cells (PSC) to obtain an expanded population of PSC; and expressing in the expanded population of PSC a protein selected from ZNF281, LHX8 and SOHLH1 to obtain oogonium-like cells.
22. The method of claim 21, wherein the expanded population PSCs comprise a designed polynucleotide comprising an open reading frame encoding ZNF281.
23. The method of claim 21, wherein the expanded population PSCs comprise a designed polynucleotide comprising an open reading frame encoding LHX8.
24. The method of claim 21, wherein the expanded population PSCs comprise a designed polynucleotide comprising an open reading frame encoding SOHLH1.
25. The method according to claim 21, wherein the PSCs of the expanded population further comprise a designed polynucleotide comprising an open reading frame encoding the FIGLA protein.
26. The method of claim 25, wherein the expanded population PSCs further comprise: a designed polynucleotide comprising an open reading frame encoding the DLX5 protein; and a designed polynucleotide comprising an open reading frame encoding the HHEX protein.
27. The method of claim 26, wherein the expanded population PSCs further comprise: an engineered polynucleotide comprising an open reading frame encoding the DDX4 protein; an engineered polynucleotide comprising an open reading frame encoding the DAZL protein; and an engineered polynucleotide comprising an open reading frame encoding the BOLL protein.
28. The method according to claim 21, wherein the open reading frame of the constructed polynucleotide is operably linked to a heterologous promoter.
29. The method according to claim 21, wherein the heterologous promoter is an inducible promoter.
30. The method according to claim 21, wherein the population contains 1⋅10 2 -1⋅10 7 PSC.
31. The method of claim 21, wherein the PSC population is cultured for about 3-5 days, optionally about 4 days.
32. The method according to claim 31, wherein the PSC population is cultured for about a day.
33. The method of claim 21, wherein the oogonial cells are DDX4 + .
34. The method of claim 21, wherein the oogonial cells independently have a diameter of about 20 μm to 180 μm.
35. A method comprising: (a) delivering to pluripotent stem cells (PSCs) an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from ZNF281, LHX8 and SOHLH1; (b) culturing the PSCs in serum-free culture medium without a feeder to obtain an expanded PSC population; and (c) culturing PSC expanded population in a series of induction media containing an inducing agent to obtain DDX4 + oogonia-like cells.
36. The method of claim 35, wherein the engineered polynucleotide is a transposon, and the delivery further comprises delivering a transposase to the PSC.
37. The method of claim 35, wherein the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.
38. The method of claim 35, wherein the serum-free feeder-free growth medium in step (b) comprises a solubilized basement membrane preparation extracted from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells.
39. The method according to claim 38, wherein the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.
40. The method of claim 39, wherein the ECM proteins are selected from laminin, collagen IV, heparan sulfate proteoglycans, and entactin / nidogen.
41. The method according to claim 35, wherein the serum-free feeder-free growth medium in step (b) comprises growth factors selected from recombinant human basic fibroblast growth factor (rh-bFGF) and recombinant human transforming growth factor β (rh-TGFβ).
42. The method of claim 35, wherein the culturing in step (b) continues for about 6 to about 24 hours.
43. The method of claim 35, wherein the PSC of the expanded population in step (c) is cultured at a density of about 2000 cells / cm 2 up to approximately 3000 cells / cm 2 .
44. The method of claim 35, wherein culturing in step (c) comprises culturing the PSC in a first induction medium, culturing the PSC in a second induction medium, culturing the PSC in a third induction medium, and culturing the PSC in a fourth induction medium.
45. The method of claim 44, wherein the first induction medium comprises one or more of B-27, L-alanyl-L-glutamine, an inducing agent (e.g., doxycycline), activin A, a glycogen synthase kinase-3 (GSK) inhibitor, and a selective inhibitor of FGFR1 and FGFR3.
46. The method of claim 44, wherein the second induction medium comprises one or more of B-27, an inducing agent (e.g., doxycycline), a small molecule inhibitor of tankyrase (TNKS), and human bone morphogenic protein 4 (hBMP4).
47. The method of claim 44, wherein the third induction medium comprises one or more of B-27, an inducing agent (e.g., doxycycline), a small molecule tankyrase inhibitor, stem cell factor (SCF), and epidermal growth factor (EGF).
48. The method of claim 44, wherein the fourth induction medium comprises one or more of B-27, an inducing agent (e.g., doxycycline), a small molecule tankyrase inhibitor, hBMP4, SCF, and EGF.
49. An oogonium-like cell obtained by the method described in any of the preceding paragraphs.