Methods for production of platelets from pluripotent stem cells and compositions thereof
View Patent ↗Methods for production of platelets from pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are provided. These methods may be performed without forming embryoid bodies or clusters of pluripotent stem cells, and may be performed without the use of stromal inducer cells. Additionally, the yield and/or purity can be greater than has been reported for prior methods of producing platelets from pluripotent stem cells. Also provided are compositions and pharmaceutical preparations comprising platelets, preferably produced from pluripotent stem cells.
1. A method for producing β2 microglobulin-deficient platelets from megakaryocytes comprising:
contacting a non-adherent feeder-free culture of β2 microglobulin-deficient megakaryocytes with hematopoietic expansion medium and
(1) thrombopoietin (TPO) or a TPO agonist, stem cell factor (SCF), interleukin (IL)-6, and IL-9; or
(2) TPO or a TPO agonist, SCF, and IL-11; or
(3) TPO or a TPO agonist
to cause the formation in culture of pro-platelets that release β2 microglobulin-deficient platelets, at least 60% of which are positive for CD41a and CD42b expression.
2. The method of claim 1 , wherein the TPO agonist comprises at least one of: ADP, epinephrine, thrombin, collagen, TPO-R agonists, TPO mimetics, second-generation thrombopoietic agents, romiplostim, eltrombopag (SB497115, Promacta), recombinant human thrombopoietin (TPO), pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), Fab 59, AMG 531, Peg-TPOmp, TPO nonpeptide mimetics, AKR-501, monoclonal TPO agonist antibodies, polyclonal TPO agonist antibodies, TPO minibodies, VB22B sc(Fv)2, domain subclass-converted TPO agonist antibodies, MA01G4G344, recombinant human thrombopoietins, recombinant TPO fusion proteins, and TPO nonpeptide mimetics.
3. The method of claim 1 , wherein the non-adherent culture of megakaryocytes is a feeder-free culture.
4. The method of claim 1 , wherein the culture is
in a medium comprising one or more of: SCF at 0.5-100 ng/ml, TPO at 10-100 ng/ml, and IL-11 at 10-100 ng/ml, at least one ROCK inhibitor, and/or Heparin at 2.5-25 Units/ml; or
in a medium comprising one or more of: TPO at 10-100 ng/ml, SCF at 0.5-100 ng/ml, IL-6 at 5-25 ng/ml, IL-9 at 5-25 ng/ml, at least one ROCK inhibitor, and/or Heparin at 2.5-25 units/ml.
5. The method of claim 4 , wherein the ROCK inhibitor comprises Y27632.
6. The method of claim 1 further comprising isolating the platelets.
7. The method of claim 1 , further comprising subjecting the megakaryocytes to a shearing force.
8. The method of claim 1 , wherein the megakaryocytes are cultured in the presence of an exogenously added protease inhibitor or an exogenously added matrix metalloprotease (MMP) inhibitor.
9. The method of claim 1 , wherein the megakaryocytes are cultured at a temperature greater than 37° C. and equal to or less than 40° C.
10. A method of producing platelets, comprising:
(a) culturing pluripotent stem cells engineered to be β2 microglobulin deficient in a feeder-free culture comprising TPO or a TPO agonist to form-megakaryocytes; and
(b) contacting a non-adherent feeder-free culture of the megakaryocytes with hematopoietic expansion medium and
(1) thrombopoietin (TPO) or a TPO agonist, stem cell factor (SCF), interleukin (IL)-6, and IL-9, or
(2) TPO or a TPO agonist, SCF, and IL-11, or
(3) TPO or a TPO agonist
to cause the formation in culture of pro-platelets that release platelets, at least 60% of which are positive for CD41a and CD42b expression.
11. The method of claim 10 , wherein a BET inhibitor is added to the culture.
12. The method of claim 10 , wherein the pluripotent stem cells are β2 microglobulin knockout pluripotent stem cells.
13. A method of producing platelets, comprising
contacting a non-adherent feeder-free culture of β2 microglobulin knockout megakaryocytes with hematopoietic expansion medium and
(1) thrombopoietin (TPO) or a TPO agonist, stem cell factor (SCF), interleukin (IL)-6, and IL-9, or
(2) TPO or a TPO agonist, SCF, and IL-11, or
(3) TPO or a TPO agonist
to cause the formation in culture of pro-platelets that release platelets, at least 60% of which are positive for CD41a and CD42b expression.
14. A method of producing platelets, comprising
contacting a non-adherent feeder-free culture of HLA-A-deficient megakaryocytes with hematopoietic expansion medium and
(1) thrombopoietin (TPO) or a TPO agonist, stem cell factor (SCF), interleukin (IL)-6, and IL-9, or
(2) TPO or a TPO agonist, SCF, and IL-11, or
(3) TPO or a TPO agonist
to cause the formation in culture of pro-platelets that release platelets, at least 60% of which are positive for CD41a and CD42b expression.