Trophoblast-derived mesenchymal stem cells (T-MSCs) produced from human embryonic stem cells, methods and uses thereof

Patent metadata

Field	Value
Publication number	US-9725698-B2
Application number	US-201314413297-A
Country	US
Kind code	B2
Filing date	Jul 11, 2013
Priority date	Jul 11, 2012
Publication date	Aug 8, 2017
Grant date	Aug 8, 2017

Abstract

The disclosure provided herein relates generally to mesenchymal-like stem cells “hES-T-MiSC” or “T-MSC” and the method of producing the stem cells. The method comprises culturing embryonic stem cells under conditions that the embryonic stem cells develop through an intermediate differentiation of trophoblasts, and culturing the differentiated trophoblasts to hES-T-MSC or T-MSC, T-MSC derived cells and cell lineages “T-MSC-DL” are also described. Disclosed also herein are solutions and pharmaceutical compositions comprising the T-MSC and/or T-MSC-DL, methods of making the T-MSC and T-MSC-DL, and methods of using the T-MSC and T-MSC-DL for treatment and prevention of diseases, specifically, T-MSC and T-MSC-DL are used as immunosuppressive agents to treat multiple sclerosis and autoimmune diseases.

First claim

The invention claimed is:

1. A method of producing human trophoblast-derived mesenchymal stem cells (T-MSCs) from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), the method comprising:
(a) culturing hESCs or iPSCs in a medium comprising bone morphogenetic protein-4 (BMP-4), and optionally a TGFβ inhibitor, for a first time period of 1 to 5 days sufficient for the hESCs or iPSCs to differentiate into trophoblast cells;
(b) dissociating the trophoblast cells into single trophoblast cells; and
(c) plating the single trophoblast cells from step (b) onto gelatin, vitronectin, laminin, fibronectin, Matrigel or collagen-coated plates, and culturing said single trophoblast cells for a second time period of 4 to 10 days in a mesenchymal stem cell (MSC) growth medium containing LIF, bFGF or PDGF, or a combination thereof, thereby producing human T-MSCs.

2. The method of claim 1 , where the TGFβ inhibitor is an SB431542, A83-01 or ALK5 inhibitor.

3. The method of claim 1 wherein, prior to step (a), hESCs are cultured by a method comprising the following steps:
(i) culturing the hESCs to about 80% confluency on Matrigel-coated plates;
(ii) dissociating the hESCs under suitable conditions;
(iii) isolating the hESCs; and
(iv) washing the hESCs.

4. The method of claim 1 , wherein the concentration of BMP4 is about 1 to about 100 ng/ml.

5. The method of claim 1 , wherein the population of human T-MSCs: (i) comprises greater than 95% of cells expressing CD73, CD90, CD105, CD146, CD166, and CD44; (ii) comprises greater than 80% of cells expressing CD13, CD29, CD54, CD49E; (iii) comprises less than 5% of cells expressing CD45, CD34, CD31 and SSEA4; (iv) expresses IL-10 and TGFβ; (v) comprises less than 2% of cells expressing IL-6, IL-12 and TNFα; and (vi) comprises less than 0.001% of cells co-expressing OCT4, NANOG, TRA-1-60 and SSEA4.

6. The method of claim 5 , wherein the human T-MSCs do not express MMP2 and RAGE.

7. The method of claim 5 , wherein the T-MSC cells have low expression of IFNγR1 and IFNγR2 compared to expression of IFNγR1 and IFNγR2 of bone marrow-derived mesenchymal stem cells (BM-MSC).

8. The method of claim 5 , wherein the human T-MSCs further express CD73 and do not express IL-6.

9. The method of claim 5 , wherein the human T-MSCs further express at least one cell marker selected from the group consisting of CD90, CD105, CD13, CD29, CD54, CD146, CD166, and CD44; do not express at least one marker selected from the group consisting of CD34, CD31, and CD45; and do not express at least one marker selected from the group consisting of MMP, RAGE, IFNγR1, IFNγR2, IL-12, TNFα and VCAM1.

10. The method of claim 5 , wherein the human T-MSCs are irradiated.

11. The method of claim 7 , wherein the human T-MSCs are irradiated with gamma-irradiation.

12. A method of producing human trophoblast-derived mesenchymal stem cells from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), the method comprising:
(a) culturing hESCs or iPSCs in a medium comprising bone morphogenetic protein-4 (BMP-4) for 1½ to 5 days for the hESC or iPSC to differentiate into trophoblast cells, and optionally with a TGFβ inhibitor;
(b) isolating the trophoblast cells;
(c) dissociating the trophoblast cells from step (b) into single trophoblast cells; and
(d) plating the single trophoblast cells from step (c) onto Matrigel-coated plates and culturing said single trophoblast cells for 4-10 days in a mesenchymal stem cell (MSC) growth medium containing serum, knockout serum replacement (KOSR), or in a serum-free medium, in an amount sufficient to induce differentiation of the single trophoblast cells into human trophoblast-derived mesenchymal stem cells; wherein at least 90% of the human trophoblast-derived mesenchymal stem cells express CD73.

Assignees

• Imstem Biotechnology Inc
• Univ Connecticut

Inventors

• Wang Xiaofang
• Xu Ren-He

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title

• A61P37/02

  Immunomodulators · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• C12N2501/145

  Thrombopoietin [TPO] · CPC title