Minimal volume reprogramming of mononuclear cells
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.
1. A composition comprising CD34+ mononuclear cells, wherein the CD34+ mononuclear cells:
(a) are from an individual segment of umbilical cord blood (UCB) comprising at least 1×10 3 mononuclear cells, wherein the individual segment has a volume of less than 500 uL; and
(b) comprise:
(i) at least one exogenous nonintegrated polynucleotide encoding one or more of octamer-binding transcription factor 4 (OCT4), sex determining region Y box 2 (SOX2) and Simian virus 40 large antigen (SV4OLT); or one or more exogenous polypeptides selected from the group consisting of an OCT4 polypeptide, a SOX2 polypeptide, and an SV4OLT polypeptide; and
(ii) CD45+/CD34+/Lineage− cells; and
wherein the CD34+ mononuclear cells are suitable for being reprogrammed into induced pluripotent stem cells (iPSCs) without increasing the volume of the individual segment to more than 500 uL and wherein the reprogrammed iPSCs co-express SSEA4 and TRA181 with reprogramming efficiency of at least 0.1% efficiency based on the number of iPSCs divided by the number of CD34+ mononuclear cells in the individual segment.
2. The composition of claim 1 , wherein the individual sample comprises about 1×10 3 to about 1×10 6 mononuclear cells.
3. The composition of claim 1 , wherein the individual segment of UCB is an individual sample of cryopreserved UCB.
4. The composition of claim 1 , wherein the reprogramming efficiency is at least 1%.
5. The composition of claim 1 , wherein the CD34+ mononuclear cells comprise the at least one exogenous polynucleotide, and wherein the at least one exogenous polynucleotide is not integrated into the genomes of the mononuclear cells.
6. The composition of claim 1 , wherein the CD34+ mononuclear cells comprise the at least one exogenous polynucleotide, and wherein the at least one exogenous polynucleotide is selected from the group consisting of a non-integrating lentivirus, an episome, mRNA, Sendai virus, miRNA, and self-replicating RNA.
7. The composition of claim 1 , wherein the composition is free of exogenous polynucleotides encoding Avian Myelocytomatosis Viral Oncogene Homolog (c-MYC) and Kruppel-like factor 4 (KLF4) and is free of exogenous c-MYC and KLF4 polypeptides.
8. The composition of claim 1 , wherein the CD34+ mononuclear cells comprise expanded or unexpanded CD45+/CD34+/Lineage− progenitor cells.
9. The composition of claim 1 , further comprising 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.
10. The composition of claim 1 , wherein the composition
(i) is free of feeder cells, or
(ii) comprises a defined culture medium.
11. The composition of claim 1 , wherein the CD34+ mononuclear cells comprise hematopoietic stem or progenitor cells.
12. The composition of claim 11 , wherein the hematopoietic stem or progenitor cells are suitable for hematopoietic reconstitution in a subject in need of cell therapy.
13. The composition of claim 1 , wherein the CD34+ mononuclear cells are
(i) allogeneic to a subject in need of cell therapy, and
(ii) HLA matched to the subject.
14. The composition of claim 1 , wherein the individual sample has a volume of about 50 μL to about 500 μL.
15. The composition of claim 1 , wherein the individual sample has 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.