Minimal volume reprogramming of mononuclear cells

Patent metadata

Field	Value
Publication number	US-10023879-B2
Application number	US-201514731403-A
Country	US
Kind code	B2
Filing date	Jun 4, 2015
Priority date	Jun 4, 2014
Publication date	Jul 17, 2018
Grant date	Jul 17, 2018

Abstract

The invention provides compositions and methods for reprogramming minimal volumes of mononuclear cells. In particular aspects, the invention provides methods and compositions for reprogramming minimal volumes of umbilical cord blood obtained from cord blood segments from cryopreserved cord blood segments.

First claim

The invention claimed is:

1. A method for producing induced pluripotent stem cells (iPSC) from an individual segment of an umbilical cord blood (UCB) unit comprising:
providing an individual segment of UCB comprising at least 1×10 3 mononuclear cells, wherein said mononuclear cells comprise CD34+ mononuclear cells comprising CD45+/CD34+/Lineage− cells, wherein the individual segment of UCB has a volume of less than 500 μL;
reprogramming by a non-integrating method said CD34+ mononuclear cells to iPSC by introducing to the CD34+ mononuclear cells a composition comprising at least one polynucleotide encoding a reprogramming factor selected from the group consisting of octamer-binding transcription factor 4 (OCT4), sex determining region Y box 2 (SOX2) and Simian virus 40 large antigen (SV40LT), wherein reprogrammed iPSC co-express SSEA4 and TRA181 with reprogramming efficiency of at least 0.1%.

2. The method of claim 1 , wherein the composition is free of polynucleotides encoding Avian Myelocytomatosis Viral Oncogene Homolog (c-MYC) and Kruppel-like factor 4 (KLF4) and is free of c-MYC and KLF4 polypeptides.

3. The method of claim 1 , wherein the individual segment comprises about 1×10 3 to about 1×10 6 mononuclear cells.

4. The method of claim 1 , wherein the mononuclear cells comprise CD45+/Lineage− progenitor cells with or without being expanded prior to reprogramming.

5. The method of claim 1 , wherein the at least one polynucleotide is selected from the group consisting of a non-integrating lentivirus, an episome, mRNA, Sendai virus, miRNA, and self-replicating RNA.

6. The method of claim 1 , wherein reprogramming further comprises contacting the mononuclear cells with at least one inhibitor selected from the group consisting of: a glycogen synthase kinase 3 (GSK3) inhibitor, a mitogen-activated protein kinase kinase enzyme (MEK) inhibitor, a rho-associated protein kinase (ROCK) inhibitor, and a transforming growth factor β receptor (TGFβR) inhibitor; and optionally, wherein reprogramming is performed in a culture environment that is free of feeder cells, or is in a defined culture medium.

7. The method of claim 1 , wherein the method further comprises culturing the iPSC after the reprogramming in a further composition comprising a Wnt pathway agonist, a mitogen-activated protein kinase kinase enzyme (MEK) inhibitor, and a rho-associated protein kinase (ROCK) inhibitor, but wherein the further composition does not comprise a transforming growth factor β receptor (TGFβR) inhibitor.

8. The method of claim 7 , wherein culturing is performed in a culture environment selected from the group consisting of a feeder-free environment, a single cell enzymatically disassociated culturing environment, and a defined culture medium environment.

9. The method of claim 7 , wherein culturing produces ground state iPSCs.

10. The method of claim 1 , wherein the efficiency of the reprogramming is at least 1%.

11. The method of claim 1 , further comprising banking the iPSC produced from the UCB.

12. The method of claim 1 , wherein the iPSCs produced from the UCB are allogeneic to a subject in need of cell therapy and are HLA matched for the subject.

13. The method of claim 1 , wherein the individual segment of UCB is cryopreserved.

14. The method of claim 1 , wherein providing an individual segment of UCB further comprises selecting CD34+ mononuclear cells from the individual segment of UCB.

15. The method of claim 14 , further comprising culturing the selected CD34+ mononuclear cells under conditions suitable to expand the CD34+ mononuclear cells.

16. The method of claim 14 , wherein selecting is accomplished by Ficoll gradient, fluorescent assisted cell sorting (FACS) or magnetic assisted cell sorting (MACS).

17. The method of claim 14 , wherein the CD34+ mononuclear cells comprise hematopoietic stem or progenitor cells.

18. The method claims of 83, wherein culturing is performed under defined culture conditions.

19. The method of claim 17 , wherein the hematopoietic stem or progenitor cells are suitable for hematopoietic reconstitution in a subject in need of cell therapy.

20. The method of claim 19 , wherein the hematopoietic stem and progenitor cells are allogeneic to the subject in need of cell therapy and HLA matched to said subject.

21. The method of claim 1 , wherein the individual segment of UCB is cryopreserved, and wherein prior to introducing the composition to the CD34+ mononuclear cells, the method comprises:
selecting CD34+ mononuclear cells from the individual segment of UCB and
culturing the CD34+ mononuclear cells under conditions suitable to expand CD34+ mononuclear cells.

22. The method of claim 1 , wherein the volume is between 50 μL and 500 μL.

23. The method of claim 1 , wherein the volume is a volume selected from the group consisting of: less than 400 μL, less than 300 μL, less than 200 μL, less than 100 μL, less than 50 μL, less than 25 μL, and less than 10 μL.

Assignees

• Fate Therapeutics Inc

Inventors

• Flynn Peter
• Valamehr Bahram

Classifications

• A61K35/51

  Umbilical cord; Umbilical cord blood; Umbilical stem cells · CPC title

• A61K35/545

  Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells · CPC title

• C12N2800/108

  episomal vectors · CPC title

• C12N15/85 Primary

  for animal cells · CPC title

• C12N2840/20

  translation of more than one cistron · CPC title