Populations of hematopoietic progenitors and methods of enriching stem cells therefor

Patent metadata

Field	Value
Publication number	US-9834754-B2
Application number	US-201214359517-A
Country	US
Kind code	B2
Filing date	Nov 21, 2012
Priority date	Nov 21, 2011
Publication date	Dec 5, 2017
Grant date	Dec 5, 2017

Abstract

There is described herein a method of enriching a population of stem cells for hematopoietic progenitors. The method comprises inducing hematopoietic differentiation in a population of human embryonic stem cells or human induced pluripotent stem cells; sorting the population based on expression of CD43 and at least one of CD34, CD31 and CD144; and selecting a fraction that is at least one of CD34+CD43−, CD31+CD43− and CD144+CD43−. Also provided are populations of hematopoietic progenitors obtained by the methods described herein.

First claim

The invention claimed is:

1. A method of enriching a population of stem cells for hematopoietic progenitors from a population of human pluripotent stem cells that have been induced to undergo hematopoietic differentiation, the method comprising:
a. inducing hematopoietic differentiation in a population of human pluripotent stem cells, wherein activin/nodal signaling is inhibited between day 1 and day 4 of differentiation;
b. sorting the induced population based on expression of CD34 and CD43; and
c. selecting a fraction of the cell population that is CD34 + CD43 − , and

wherein the sorting and cell fraction selection is performed on a day selected from about day 6 to about day 11 of differentiation.

2. The method of claim 1 , wherein inducing hematopoietic differentiation is conducted serum free.

3. The method of claim 1 , wherein inducing hematopoietic differentiation is conducted stroma free.

4. The method of claim 1 , wherein inducing hematopoietic differentiation comprises culturing the population with BMP4.

5. The method of claim 1 , wherein inducing hematopoietic differentiation comprises culturing the population with bFGF.

6. The method of claim 1 , wherein inducing hematopoietic differentiation comprises culturing the population with at least one of VEGF, DKK, IL-6 and IL-11, and/or combinations thereof, between days 3 and 11, or between days 4 and 9.

7. The method of claim 1 , wherein inducing hematopoietic differentiation comprises culturing the population with at least one of SCF and EPO, between days 4 and 11, or between days 6 and 9.

8. The method of claim 1 , wherein inducing hematopoietic differentiation comprises culturing the population with at least one of TPO, Flt-3 and IL-3, and/or combinations thereof, between days 6 and 11, or and 8 and 9.

9. The method of claim 1 , wherein inhibiting activin/nodal signaling comprises culturing the population with an activin/nodal inhibitor.

10. The method of claim 9 , wherein the inhibitor is SB-431542.

11. The method of claim 1 , wherein the fraction comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% hematopoietic progenitors.

12. The method of claim 10 , wherein the population is cultured with between 1 μM to 10 mM, 1 μM to 1 mM, or about 6 μM SB-431542.

13. The method of claim 1 , wherein activin/nodal signaling is inhibited between day 2 and day 4 of differentiation.

14. The method of claim 13 , wherein activin/nodal signaling is inhibited by culturing the population is cultured with between 1 μM to 10 mM, 1 μM to 1 mM, or about 6 μM SB-431542.

Assignees

• Univ Health Network
• Sunnybrook Res Inst

Inventors

• Keller Gordon
• Zuniga-Pflucker Juan Carlos
• Kennedy Marion J
• Sturgeon Christopher Michael
• Ditadi Andrea
• Awong Geneve Sheandra

Classifications

• A61P7/06

  Antianaemics · CPC title

• C12N2501/165

  Vascular endothelial growth factor [VEGF] · CPC title

• C12N2501/2307

  Interleukin-7 (IL-7) · CPC title

• G01N33/56972

  White blood cells · CPC title

• C12N2501/155

  Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor · CPC title