# US 2019/0167733 A1 · Mesenchymal-like stem cells derived from human embryonic stem cells, methods and uses thereof

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- Number: US 2019/0167733 A1
- Document ID: US20190167733A1
- Status: Published application
- Kind: A1
- Application number: 16/254,986
- Filed: 2019-01-23
- Published: 2019-06-06
- Earliest claimed: 2012-07-11
- Assignees: Imstem Biotechnology, Inc.; University of Connecticut
- Applicants: Imstem Biotechnology, Inc.; University of Connecticut
- Inventors: Xiaofang WANG; Ren-He XU
- Web page: https://patentgrep.com/patent/US20190167733A1

## CPC

- A61K 35/545: Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 17/00: Drugs for dermatological disorders
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 19/04: for non-specific disorders of the connective tissue
- A61P 19/08: for bone diseases, e.g. rachitism, Paget's disease
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 5/0668: {Mesenchymal stem cells from other natural sources}
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/135: Platelet-derived growth factor [PDGF]
- C12N 2501/145: Thrombopoietin [TPO]
- C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
- C12N 2501/165: Vascular endothelial growth factor [VEGF]
- C12N 2501/235: Leukemia inhibitory factor [LIF]
- C12N 2501/26: Flt-3 ligand (CD135L, flk-2 ligand)
- C12N 2506/02: from embryonic cells
- C12N 2506/45: from artificially induced pluripotent stem cells
- C12N 2529/00: Culture process characterised by the use of electromagnetic stimulation
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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 trophoblast in 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.

## Claims

### Claim at position 1

1-133. (canceled)

### Claim 134

A population of trophoblast-derived MSCs (T-MSCs), adipocytes, chondrocytes, osteoblast cells, neural lineage cells, myoblast cells, stromal cells and fibroblast cells produced by the method comprising:
a. culturing human embryonic stem cells in a serum free medium comprising at least one GSK3 inhibitor at a concentration ranging from 0.05 μM to 0.2 μM, wherein the human embryonic stem cells are cultured in the absence of feeder cells;
b. culturing the cells from step a) in a serum-free medium comprising vascular endothelial growth factor (VEGF) and bone morphogenic protein 4 (BMP4) in an amount sufficient to induce formation of embryoid bodies comprising human hemangio-colony forming cells;
c. adding at least one growth factor to the culture resulting from step b), wherein the growth factor is in an amount sufficient to expand human hemangio-colony forming cells;
d. disaggregating the hemangio-colony forming cells resulting from step c) into single cells; and
e. culturing the single hemangio-colony forming cells resulting from step d) in mesenchymal stem cell medium containing serum, knockout serum replacement (KOSR), or in a serum-free medium to induce differentiation of the single cells into human mesenchymal stem cells;
wherein at least 90% of the hES-MSCs express CD73, and said hES-MSCs comprise greater than 95% of cells expressing CD73, CD90, CD105, CD146, CD166, and CD44; (ii) comprise greater than 80% of cells expressing CD13, CD29, CD54, and CD49E; (iii) comprise less than 5% of cells expressing CD45, CD34, CD31 and SSEA4; (iv) express IL-10 and TGFβ; (v) comprise less than 2% of cells expressing IL-6, IL-12 and TNFα; and (vi) comprise less than 0.001% of cells co-expressing OCT4, NANOG, TRA-1-60 and SSEA4.

### Claim 135 (depends on 134)

A pharmaceutical composition comprising the population of T-MSCs of claim 134 and a pharmaceutically acceptable carrier.

### Claim 141 (depends on 134)

A method for treating a T cell, B cell, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the population of T-MSCs of claim 134 in an amount sufficient to ameliorate or alleviate at least one symptom of the diseases, reverse the diseases, minimize the extent of the disease or hinder its development.

### Claim 142 (depends on 134)

A kit comprising the population of T-MSCs of claim 134 and a carrier.

### Claim 143 (depends on 142)

The kit of claim 142 further comprising a thawing reagent, immunosuppressive enhancer, and anti-histamine.

### Claim 144 (depends on 134)

A method of delivering an agent through the blood-brain barrier and/or the blood-spinal cord barrier, said method comprising the steps of: attaching the agent to a hES-MSC from the population of hES-MSC of claim 134 to form a hES-MSC-agent complex; and administering the hES-MSC-agent complex to a subject in need thereof, wherein the hES-MSC is capable of crossing the blood-brain barrier and/or the blood-spinal cord barrier and the agent is for the treatment, prevention or diagnosis of a disease or injury in the subject in need thereof.

### Claim 145 (depends on 134)

A method for preventing or treating a T cell and/or B cell related autoimmune disease comprising administering to a subject in need thereof the population of hES-MSC of claim 134, an amount sufficient to ameliorate or alleviate at least one symptom of the T cell related autoimmune disease or reverse the T cell and /or B cell related autoimmune disease.

### Claim 146 (depends on 134)

A method for preventing or treating a multiple sclerosis comprising administering to a subject in need thereof the population of hES-MSC of claim 134, an amount sufficient to ameliorate or alleviate at least one symptom of the disease or reverse the disease.

### Claim 147 (depends on 134)

A method for tissue regeneration or repair comprising administering to a subject in need thereof the population of T-MSCs of claim 134 in an amount sufficient to promote tissue regenerations including, but not limited to, joint regeneration, tendon regeneration, connective tissue regeneration, neural lineage cells regeneration, fat tissue regeneration, bone regeneration, skin regeneration, muscle regeneration, cartilage regeneration, smooth muscle regeneration, cardiac muscle regeneration, epithelia tissue regeneration, and/or ligament regeneration.