WO2014011881A2

WO2014011881A2 - Mesenchymal-like stem cells derived from human embryonic stem cells, methods and uses thereof

Info

Publication number: WO2014011881A2
Application number: PCT/US2013/050077
Authority: WO
Inventors: Xiaofang Wang; Ren-He Xu
Original assignee: Imstem Biotechnology, Inc.
Priority date: 2012-07-11
Filing date: 2013-07-11
Publication date: 2014-01-16
Also published as: CA2876512A1; US20170290864A1; JP6277187B2; AU2013290146A1; US20150191699A1; WO2014011881A3; EP2872619A4; US9725698B2; CA3176706A1; US10842826B2; AU2013290146B2; JP2015523083A; CN104487568A; EP2872619A2; CA2876512C; CN104487568B; EP2872619B1; HK1208055A1; US20210085725A1; US10226488B2

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 pharmaceutica! 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.

Description

MESENCHYMAL -LI E STEM CELLS DERIVED FROM HUMAN EMBRYONIC STEM CELLS,

METHODS AND USES THEREOF

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Application Serial No.

61 /870,192, filed July 1 1 , 2012 and U.S. Provisional Application Serial No. 61/684,509, filed August 17, 2012 _; which are hereby incorporated by reference in their entireties.

1. INTRODUCTION

The disclosure provided herein relates generally to mesenc rymaS-fike stem cells ''hES-T- fvISC" or "T-MSC^S and the method of producing the stem cells. The method comprises cuSturing embryonic stem cells under conditions that the embryonic stem cells develop through an intermediate differentiation of trophoblasts, and differentiating trophoblasts into hES-T-MSC or T- MSC. Disclosed herein are the T-MSC, solutions and pharmaceutical compositions comprising: the T-MSC, methods of making the T-MSC, methods of using the T-MSC for treatment and prevention of diseases, specifically, T-MSC are used as an immunosuppressive agent to treat multiple sclerosis and other autoimmune diseases, for tissue regeneration/repair uses, and methods of using the T-MSC for the delivery of agents across the blood brain barrier and the blood spinal cord barrier. Also disclosed herein are methods of using T-MSCs to modulate the immune system, inhibit immune response to an individual's seif-antigen and repair damaged central nervous systems. Compositions comprising T-MSCs for us in immunomoduSation are disclosed herein, as are methods of providing modified T-MSC with improved immunosuppressive function through modified gene expression. 2. BACKGROUND

Human mesenchymal stem/siroma! ceils ( SCs) have been widely used for immune system regulation and tissue repair. Human embryonic stem cells (hESCs) can be used as a reliable source for generating high-quality human MSCs. There are many methods to differentiate hESCs into MSCs. However, current methods are not able to conduct such differentiation in an efficient manner to produce a high yield of high purity MSCs,

Mesenchymal stem cells {MSCs} derived from adult mouse or human tissues such as bone marrow, umbilical cord and fat tissue are multipotent i.e., capable of generating a variety of mature cell lineages including adipocytes, chondrocytes, osteoblast cells, neural lineage cells, myoblast, stroma! cells and fibroblast, etc. These technologies have been well characterized and patented. For example, see Caplan et aL U.S. Pat. No, 5,486,359 (human mesenchymal stem cells). However, the currently available adult tissue-derived MSCs have several pitfalls. First, the limited sources and varying quaiity of the donor tissues suc as the bone marrow restrict the study and application of the SCs and prevent the standardization of the MSCs as a medical product for large-scale clinical use. Second, the MSCs obtained from the aduit tissues are highly mixed populations of ceils, in which only a small portion of the cells have strong immunosuppressive effect. To obtain enough ceil numbers for clinical use, in vitro expansion is necessary, which can decrease the immunosuppressive and homing abilities of MSCs (Javazon et at, 2004). Third, there are safety issues regarding to the use of adult-derived MSCs including malignant transformation (Wong, 2011 } and potential transmission of infectious pathogens from donors.

To overcome these pitfalls, scientists have attempted to derive MSCs from hESCs via various methods. These methods involve either co-culture with the mouse OP9 cell line or handpicking plus the use of multiple cytokines and chemicals (Barberi et at, 2005; Chen et at, 2012; Liu et at, 2012; Sanchez et al„ 201 1 }. Recently, a TGFp signaling inhibitor SB431542 has been used to differentiate hESCs into MSCs, which simplifies the procedures and improves the efficiency (Chen et at, 2012), but the yield and purity are quite low (see the below-described comparison tests.). In 2010, the inventors and Advanced Cell Technology developed another method to derive MSG from hemangioblast, which involved the use of many expensive cytokines and methylcellulose medium, but the derivation efficiency is also low using this method.

Currently known methods for differentiation of hESCs into MSCs are each characterized as having one or more serious shortcomings and weaknesses; Differentiation of MSCs from hESCs co-cultured with the OP9 stroma! cells has the disadvantages of being time consuming, producing cells of low yield, low purity, and using animal feeder cells and undefined culture conditions {Barberi et a!,, 2005). Differentiation from outgrowing cells around rep!ated embryoid bodies formed by hESCs has the disadvantages of being time consuming, producing ceils in low yield, using undefined culture condition, and being an expensive method (Olivier et at, 2006). Differentiation from hESCs cultured on collagen-coated plates has the disadvantages of very low yield, undefined culture conditions, and being time consuming (Liu et at, 2012), Differentiation with hESCs treated with inhibitors of TGFfS signaling has the disadvantages including low purity of cells (per our tests), low cell yield, time consuming method, and low immunosuppressive effect of the ceils that are produced (Chen et at. , 2012; Sanchez ei a/,, 2011 ). Thus, there is a need for an unlimited, safe, highly stable, efficient and consistent source of MSCs to use as a treatment and prophylactic for various diseases.

Multiple sclerosis (MS) is a chronic autoimmune diseas caused by infiltration of peripheral immune cells into the centra! nervous system (CNS) through damaged blood-brain barrier (B8B) or biood-spinal cord barrier (BSCS), which causes inflammation of the myelin sheaths around neuronal axons, and causes demye!ination and scarring of the axons (McFarSand and Martin (2007)). According to the National Multiple Sclerosis Society of United States, there are more than 70 FDA-approved medications for the treatment of MS, including Avonex (ΙΡΝβ- 1 a), Betaseron (iFNp-l b), Giienya (a sphingosine 1- phosphate receptor modulator), Glatiramer acetate (or Copolymer 1 ), and Tysabri (humanized anfi-a-integrin antibody). However, these offer only palliative relief and are associated with serious adverse effects including increased infection, heart attack, stroke, progressive multifocal ieukoencephaiopathy, arrhythmia, pain, depression, fatigue, macula edema, and erectile dysfunction (Johnston and So (2012); Weber et al. (2012)).

Transplantation of mesenchymal stromal/siem cells (MSCs) has emerged as a potentially attractive therapy due to their immunomodulatory and neuroregenerative effects (Auietta et a ., (20 2); Pitteoger et at. (1933)) and potential ability to repair the blood-brain barrier (Chao et a/.

(2009) ; Menge et a/. (2012)), MSCs are muittpotent meaning they can generate a variety of cell lineages including adipocyte, chondrocyte, osteoblast cells and neurons. They can be derived from fetal, neonatal, and adult tissues such as the amniotic membrane, umbilical cord, bone marrow, and adipose. MSCs have several unique advantages over current pharmacotherapies, as these cells can serve as carriers of multiple and potentially synergistic therapeutic factors, and can migrate to injured tissues to exert local effects through secretion of mediators and cel!-ce!! contact (Uccellf and Prockop (2010a)). Importantly, MSCs have been found efficacious in the treatment of mice with experimental autoimmune encephalomyelitis (EAE), a well -recognized animal model of MS (Gordon et at:, 2008a; Gordon et at. (2010); Morando et at. (2012); Peron et at. (2012); Zappia et at. (2005); Zhang et at. (2005)), as well as MS patients in clinical tria!s (Connick et at. (2012); arussis et at. (2010); ohyeddin Bonab et at. (2007); Yamouf et a/.

(2010) ). Xenogeneity does not appear problematic as both mouse and human bone marrow- derived MSC (BM-MSC) can attenuate disease progression of EAE mice (Gordon et at. (2008a); Gordon et at. (2010); Morando et aL (2012); Peron et at. (2012); Zappia et aL (2005); Zhang ei a/. (2005)). However, varying effects were reported on EAE mice treated with BM-MSC in different reports (Gordon et al. (2008a); Payne et aL (2012); Zappia et aL (2005); Zhang et al. (2005)). The efficacy of BM-MSC on treatment of the disease is questionable.

There is a strong need for an unlimited, safe, highly stable, efficient and consistent source of MSC to use as a treatment and prophylactic for these diseases as well as others. Disclosed herein are hES-T-MSCs derived from hESCs through a highly efficient differentiation method that meets these needs. Also disclosed herein are a microarray analysis and other analysis, where several key factors are identified that are differentially expressed in hES-T-MSC compared to BM-MSC and other hES- SC differentiated through other methods. 3, SUMMARY

Disclosed herein is a method to derive mesenchymal-! ike stem cells from hESCs through an intermediate step of trophob!ast induction. The MSCs derived via this method ar called "hES-T-MSC^* or "T-MSC. The T-MSC may be differentiated into cells or cell lineages including, but not limited to, adipocytes, myoblast cells, neuron ceils, osteoblast cells, fibroblast, chondrocytes, stromal cells The T-MSC derived cells or ceil lineages or called "T-MSC derived lineages³ or T~MSC~DL".

Disclosed herein are compositions, including compositions comprising T-MSC and/or T-

MSC-DL, having immunosuppressive properties. Described herein are populations of T-MSC and/or T-MSC-DL selected on the basis of their ability to modulate an immune response, and compositions having immunomodulatory properties. As disclosed herein, T-MSC and/or T-MSC- DL have higher immunosuppressive activity compared to bone marrow-derived MSCs.

Disclosed herein is a method to efficiently produce T-MSC in high purity and high yield.

The method has the features of relatively few steps and fewer required differentiation factors than previously reported.

Disclosed herein are methods of using human embryonic stem cells (hESCs) to derive mesenchyma!-!ike stem cells through an intermediate differentiation of trophob!asts. The MSCs derived from trophob!asts are called hES-T-MSC or T-MSC. The T-MSC can be used to modulate the immune system. For example, they are effective in treating multiple sclerosis by preventing immune cell-caused damage in the centra! nervous systems.

Disclosed herein are human embryonic-derived mesenchymal stem cells produced by the methods disclosed herein.

Disclosed herein are methods to induc differentiation of T-MSC into T-MSC-DL.

Also disclosed herein is the application of the T-MSC and/or T-MSC-DL to treat multiple sclerosis and other autoimmune diseases in mammals and especially in human subjects,

it is a furthe object of the disclosed invention to provide a cell product T-MSC for use in immunornoduiation, for example, for prevention or inhibition of immunorejection during tissue or organ transplantation. In another specific embodiment of the method of reducing or suppressing an immune response, the immune response is graft-versus-host disease, in another specific embodiment, the immune response is an autoimmune disease, e.g., diabetes, lupus erythematosus, or rheumatoid arthritis.

it is a further object of the disclosed invention to provide a cell product T-MSC-DL for use in treatment of neural diseases.

The method can employ as many stem ceils provided herein as are required to effect a detectable suppression of an immune response. For example, the plurality of stem cells provided herein used to contact the plurality of immune cells can comprise 1 x 10^s T-MSC, 1 x 10⁵ T-MSC, 1 x 10^? T-MSC, 1 x 10^e T-MSC or more.

In one embodiment, the method described herein is a novel process for deriving {also referred to herein as producing) MSCs from hESCs, The method comprising the steps of: a. Culturing a cell culture comprising human embryonic stem cells in serum- free medium in the present of at least one growth factor in an amount sufficient to induce the differentiation of the embryonic stem ceils to differentiate into trophoblasts; in an embodiment, the time period of the differentiation into trophoblasts is about 2-5 days; in an embodiment, the medium comprises BMP4, with or without the presence of a TGPp inhibitor (i.e., SS431542, A83-G1 or ALK5 inhibitor, etc.) to increase the differentiation efficiency;

b. Adding at least one growth factor to the culture comprising the trophoblasts and continuing to culture in serum-free medium, wherein the growth factor is in an amount sufficient to expand the trophoblasts, in an embodiment, the medium comprises B P4 (this step is optional);

c. isolating the trophoblasts and re-plating the trophoblasts onto gelatin, iaminin, fibronectin, vitronectin, collagen or Matrigei-coated plates and cultured in a serum-containing or serum-free media in an amount sufficient to differentiate the trophoblasts into T-fvISC through pre-T-MSC, in an embodiment, the isolated trophoblasts are cultured for 4-10 days to produce the T-fvISC, wherein at least about 90%, 95%, 96%, 97%, 98%, 99% of the resulting T-MSC express cell surface markers for adult MSCs, in an embodiment, the medium comprises LIF, bFGF. or PDGF to increase expansion efficiency.

In a specific embodiment, the trophoblasts derived from hESC express Trop-2, but not

CD73,

in a specific embodiment, the pre-T-fvISC express Trop-2 and/or CD73.

In a specific embodiment, the T-MSC express CD73⁺CD105*0090*. It is an object of the disclosed method to differentiate hESCs into MSCs of high purity. In a preferred embodiment, CD73^tCD105^tCD90ⁱ T-MSC are produced with greater than 90%, 95%, 96%, 97%, 98%, 99% purity.

A large number of T-MSC with high purity is demonstrated by the observation that high percentages of the MSCs express cell-surface markers for adult MSCs. Th MSCs have higher immunosuppressive effect both in vitro and in vivo than MSCs obtained via other methods. The MSCs derived via this currently disclosed method are named hES-trophoblast-derived MSCs and are more briefly referred to herein as T-MSC.

In certain embodiments, the serum-containing medium contains fetal calf serum or human AB serum, L-glutamine and the serum-free medium contains knockout serum replacement (KOSR) or bovine serum albumin (BSA).

In certain embodiments, there is an additional step of irradiating the resulting T-MSC with gamma radiation ranging from 1gy to 200gy. in a further embodiment of the current invention, the method for generating and expanding T-MSC results in at least 10,000 T- SC, at least 50,000 T-MSC, at least 100,000 T- MSC, at least 500,000 T-MSC, at least 1 x 10⁶ T-MSC, at least 5 x 10^δ T-MSC, at least 1 x 10^? T- MSC, at least 5 x 10⁷ T-MSC, at least 1 x 10⁸ T-MSC, at least 5 x 10^s T-MSC, at least 1 x 10^s T- MSC, at ieast 5 x 10⁹ T-MSC, or at ieast 1 x 10¹⁰ T-MSC. These methods result in eel! solutions that may comprise between 10,000 and 10 billion T-MSC. in certain embodiments, at least about 90%, 91 %, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or 99% of the resulting human embryonic- mesenchymai stem cells express one or more hES-MSC differential markers. In certain embodiments, the marker is CD73, CD9Q and CD 105.

in one embodiment, the T-MSCs remarkably attenuate the disease score of the EAE mice, accompanied by decreased demyeiination, T cell infiltration, and microglial responses, in addition, the T-MSCs have much stronger immunosuppressive activity in vivo and in vitro when compared to bone marrow derived MSCs (BM-MSC). Also provided herein are key proteins/molecules that are differentia!!y expressed between T-MSC and BM-MSCs. Provided herein are methods of identifying T-MSCs with improved immunosuppressiv activity by measuring the expression level of the protein/molecular markers. Also disclosed are methods of genetic modification to improve immunosuppressive activity of T-MSCs.

A further embodiment of the present invention is a solution comprising T-MSC comprising at least 10,000 T-MSC, at least 50,000 T-MSC, at least 100,000 T-MSC, at least 500,000 T- MSC, at least 1 x 10^e T-MSC, at least 5 x 10^s T-MSC, at ieast 1 x 10⁷ T-MSC, at least 5 x 10⁷ T- MSC, at least 1 x 10^s T-MSC, at least 5 x 10^s T-MSC, at least 1 x 1Q⁹ T-MSC, at least 5 x 10^s T- MSC, or at least 1 x 10¹⁰ T-MSC.

In certain embodiments, the culture volume is from 2ml for at ieast 10,000 cells, 10ml for at Ieast 100,000 cells, 100ml for at Ieast 1 ,000,000 cells, 1000 ml for at least 10,000,000 cells, and up to 4000 ml of media for 5 x 10⁸ cells.

These solutions can be injected into a subject. These solutions can be frozen. These solutions can be used for the manufacture of a medicament for a disease that can be treated by the administration of T-MSC.

This invention aSso provides a method for producing a solution of T-MSC suitable for injection into a patient comprising the steps of isolating the solution of cells described in the preceding paragraph and placing the cells into solution suitable for injection into a patient. This invention also provides a method of producing a solution of T-MSC suitable for freezing comprising the steps of isolating the celis described in the preceding paragraph and placing into a solution suitable for freezing.

Yet another embodiment of the present invention is a T-MSC expressing one or more of eel! marker proteins including CD73, CD90, CD105, CD13, CD29, CD54, CD44, CD146, CD166 or a combination thereof. In a further embodiment, the human embryonic-mesenchyma! stem cell does not express or expresses low levels of one or more cell marker proteins including CD34, CD31 , CD45 or a combination thereof. In a furiher embodiment, the human ernbryonic- mesenchymai stem ceil does not express or expresses iow levels of one o more proinflammatory proteins including MMP2, RAGE, SFNyRl !FNvR2, IL-12, TNFa, IL-6, VCAM1 or a combination thereof, in certain embodiments, the human embryonic-mesenchymal stem ceil expressed at least half of the ievei of the above markers as compared to bone marrow derived MSC.

A further embodiment of the present invention is a ceil cuiture comprising T-MSC expressing one or more of ceil marker proteins including CD73, CD90, CD105, CD13, CD29, CD54, CD144, CD146 and CD44. in a further embodiment, the T-MSC in the cell culture do not express or express low levels of one or more cell marker proteins including CD34, CD31 and CD45. in a further embodiment, the T-MSC in the eel culture do not express or express low ieveis of one or more pro-inflammatory proteins including MMP2, RAGE, !FNyRI , IFNYR2_s IL-12, TNFa, IL-6, and VCAM1.

In certain embodiments, the ceil culture comprises at least 1 x 10^δ T-MSC, at least 1 x

10⁷ T-MSC at least 1 x 10⁸ T-MSC, at least 1 x 1Q⁹ T-MSC, or al least 1 x 10¹⁰ T-MSC.

In further embodiments, at least about 90% of the T-MSC in the cell culture express the CD73 protein, at least mor than 90% of the T-MSC express the CD73 protein, at least about 95% T~MSC express the CD73 protein, or more than 95% T-MSC express the CD73 protein. In further embodiments, at least about 96% of the T-MSC in the cell cuiture express the CD73 protein, at ieast more than 97% of the T-MSC express the CD73 protein, at least about 98% T- MSC express the CD73 protein, or more than 99% T-MSC express the CD73 protein.

in furthe embodiments, at Ieast about 75%, 80%, 85%, 90%, 95%, 99% of the T-MSC in the cell culture express at least one ceil marker protein selected from the group consisting of CD90, CD105, CD44, and CD29.

In further embodiments, at least about 80%, 85%, 90%, 95%, 99% of th T-MSG in the eel! culture do not express or express iow levels of at ieast one ce!i marker including CD34, CD31 , and CD45,

In further embodiments, at least about 75%, 80%, 85%, 90%, 95%, 99% of the T-MSC in the eel! culture do not express or express low levels of at least one pro-inflammatory protein inc!uding MMP2, RAGE, IFNyRI , !FNyR2, IL-12, TNFa, IL-6, and VCAM1. In certain embodiments, the T-MSC express high levels of CD24, TGFp2 or both.

In certain embodiments of the T-MSC or cell cultures described herein, the celis are irradiated using gamma radiation.

Further embodiments of the present invention are pharmaceutical preparations comprising; any one of the T-MSC or cell cultures described herein and pharmaceutically acceptable carriers. Yet further embodiments of the present invention are cryopreserved preparations of any of the T-MSC or cell cultures described herein.

Provided herein are methods of treating or preventing a T cell related autoimmune disease in a subject in need thereof, comprising the steps of administering a therapeutically effective amount of solution, cell culture or pharmaceutical preparation comprising T-MSC as described in the preceding paragraphs, to the subject in need thereof. The T ceil related autoimmune diseases include but are not limited to Crohn's disease, inflammatory bowel disease, graft versus host disease, systemic lupus erythematosus, and rheumatoid arthritis, T ce!i mediated delayed type hypersensitivity (Type IV hypersensitivity) i.e., Type 1 diabetes meSfitus, MS, RA, Hashimoto's thyroiditis, Crohn's, contact dermatitis, Scleroderma, etc.

In certain embodiments, the subject is preferably a mammal or avian, and most preferably human, !n certain embodiments, the solution, cell culture or pharmaceutical preparation comprises irradiated or non -irradiated T-MSC,

In certain embodiments, the method for treating or preventing disease includes combination therapy with one or more therapeutic agents for the treatment or prevention of disease.

In othe certain embodiments, the present invention provides methods fo treating or preventing multiple sclerosis disease in a subject in need thereof, comprising the steps of administering a therapeutically effective amount of solution, cell culture or pharmaceutical preparation comprising T-MSC as described in the preceding paragraphs, to the subject in need thereof. The multiple sclerosis can be relapsing/remitting multiple sclerosis, progressive/relapsing multiple sclerosis, primary multiple sclerosis, or secondary multiple sclerosis. The subject is preferably a mammal, and most preferably human. The solution, cell culture or pharmaceutical preparation can comprise irradiated or non-irradiated T-MSC.

The method can further comprise the administration of additional therapeutic agents to the subject, including but not limited to, fingoSimod, adrenocorticotropic hormone (ACTH), methylprednisoSone, dexamethasone, IFNp-l a, !FN-l b, giiairiamer acetate, cyclophosphamide, methotrexate, azathioprine, cladribine, cyclosporins, mitoxantrone, and sulfasalazine. In yet another embodiment, one or more of these therapeutic agents can be attached to the T-MSCs in order to cross the blood-brain and/or blood-spinal cord barrier, for delivery of the therapeutic agent to the central nervous system.

Provided herein is a method of delivering an agent through the blood-brain barrier and/or the blood-spinal cord barrier, the method comprising the steps of attaching or conjugating the agent to a T-MSC to form a complex; and administering the human embryonic-mesenchymai stem cell-agent complex to a subject in need thereof, wherein the T-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. T-MSC may be in the

S form of a single celt, a celi culture, a solution or a pharmaceutical preparation, Agents would inciude, but are not iimiied to, drugs, proteins, DMA, RNA, and small molecules.

A further embodiment is a delivery system comprising a T-MSC and a conjugated or attached agent, for crossing the blood-brain barrie and/or the bSood-spioai cord barrier,

The method described herein has a number of advantages. It is an object of the disclosed method to differentiate hESCs via an intermediate stage of trophobiasts, which is different from all the existing methods and leads to the following advantages.

Provided herein is a method of selecting clinical grade T-MSC for the treatment of autoimmune diseases, the T-MSC having the following characteristics; (i) contain >95% of cells expressing group- 1 markers; (ii) contain >80% of cells expressing group 2 markers; (fit) contain <5% of cells expressing group-3 markers; (iv) express IL-10 and ΤΘΡβ; (v) contain <2% of ceils expressing IL-6, IL-12 and TNFa; (vi) express high level of CXCR7, CXC12, CXCL12 but a low ievei of HOXB2, HOXB3, HOXB5, HOXB7, HOXB9, HOXA5, HOXA9 and other HOX family- genes (vii) contain <0.001 % of celis co-expressing ail group-4 markers_! wherein group-1 markers are CD73, CD90, CD105, CD146, CD166, and CD44, group-2 markers are CD13, CD29, CD54, CD49E, group-3 markers are CD45, CD34, CD31 and SSEA4, and group-4 markers are OCT4, NANOG, TRA-1-60 and SSEA4.

Provided herein is a method of modifying T-MSC to produce a population of modified MSG having the following characteristics: (i) contain >95% of cells expressing group-1 markers; (ii) contain >80% of cells expressing group 2 markers; (iii) contain <5% of celis expressing group- 3 markers (iv) expressing iL-10 and ΤΟΡ ; (v) contain <2% of ceils expressing 11-6, 11-12 and TNFa,; and (vi) contains <0.001 % of cells co-expressing ali group-4 markers, wherein group-1 markers are CD73, CD90, CD105, CD146, CD166, and CD44, group-2 markers are CD13, CD29, CD54, CD49E, group-3 markers are CD45, CD34, CD31 and SSEA4, and group-4 markers are OCT4, NANOG, TRA-1-60 and SSEA4.

Provided herein are conditioned medium, concentrat of conditioned medium, eel! iysat or other derivatives thereof that comprises one or more biomolecuies secreted by the T-MSC as described.

Provided herein is a method of using T-MSC as described herein as feeder cells for bone marrow hematopoietic stem ceil expansion and umbilicai-cord hematopoietic stem ceil expansion. In certain embodiments, the T-MSC suitable for the disclosed method express StroS. In certain embodiments, T-MSC is co-cultured with bone marrow hematopoietic stem cells and/or umbilicai-cord hematopoietic stem cells. In certain embodiments, the T-MSC are mesenchymal stromal cells. Provided herein is a co-culture of T-MSC as described herein and bone marrow hematopoietic stem ceils. Provided herein is a co-culture of T-MSC as described herein and umbilicai-cord hematopoietic stem cells. Also disclosed are kits comprising T-MSC described herein, in certain embodiments, the kits comprise T- SC and a celi delivery carrier.

to one aspect, provided herein is a method of suppressing or reducing an immune response comprising contacting a plurality of immune cells with a plurality of T-MSC for a time sufficient for the T-MSC to detectably suppress an immune response, wherein the T-MSC detectabiy suppress T ceil proliferation and/or differentiation in a mixed lymphocyte reaction (MLR) assay, in another specific embodiment, the contacting is performed in vitro, in another specific embodiment, the contacting is performed in vivo, in a more specific embodiment, the in vivo contacting; is performed in a mammalian subject, e.g., a human subject. In another more specific embodiment, the contacting comprises administering the T-MSC intravenously, intramuscularly, or into an organ in the subject (e.g., a pancreas).

Provided herein are methods of producing ceil populations comprising T-MSC selected on the basis of their ability to modulate (e.g., suppress) an immune response, in one embodiment, for example, the invention provides a method of selecting a T-MSC population comprising (a) assaying a plurality of T-MSC in a mixed lymphocyte reaction (MLR) assay; and (b) selecting the plurality of T-MSC if the plurality of T-MSC detectably suppresses CD4⁺ or CDS' T ceil proliferation in an MLR (mixed lymphocyte reaction), wherein the T-MSC express CD73, CD90, CD105, CD13, CD29, CD54, CD 44, In one embodiment, the T-MSC do not express or express at low level CD34, CD31 and CD45. In one embodiment, the T-MSC do not express or express at low level MMP2, RAGE, IFNGR2, IL-12A, !L-6 and VCAM1 ,

Provided herein are methods to differentiate T-MSC info multiple other eel! lineages including, but not limited to, adipocytes, myoblast ceils, neural Sineage ceils, osteoblast cells, fibroblast, chondrocytes, and stroma! cells.

Provided herein are methods for using T-MSC and its differentiated cellular products for tissue regeneration and/or tissue repair comprising administering T-MSC and/or T-MSC derived other cell lineages, in an amount sufficient to promote tissue regeneration 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, ligament regeneration, etc.

!n specific embodiments, the T cells and the T-MSC are present in the MLR at a ratio of, e.g., about 20:1 , 15:1 , 10:1 , 5:1 , 2:2, 1 :1 , 1 :2, 1 :5, 1 :10 or 1 :20, preferably 10:1.

It is a further object of the disclosed method to efficiently generate large numbers of MSCs via a high yield process. The disclosed method can generate about 10-fold higher numbers of MSCs compared to the starting number of hESCs. There is very little cell loss when hESCs are differentiated through the trophobiast stage, whereas, other methods usually have over 90% toss of the starting cells during the initial differentiation step, resulting in much lower ceil yields than the method disclosed herein.

it is an object of the disclosed method to provide a method that can produce SVISCs in a relatively short time. The entire process disclosed herein can be completed in no more than 6-14 days, depending on the starting hES lines.

it is an object of the disclosed method to provide a method that is low in cost. The differentiation method described herein only requires a very small amount of culture medium, and the method only requires one cytokine - BMP4, which is used in the disclosed method at a low dose.

it is an object of the disclosed method to provide a method that is low in cost. The differentiation method described herein only requires a very small amount of culture medium, and the method only requires one cytokine - Bfv1P4 and/or a TGPp inhibitor {i.e., SB431542, A83-01 or ALK5 inhibitor etc.).

It is an object of the disclosed method to provide a method that is high in yield. The differentiation method described herein can produce 1-5 x 10^!0 T-MSC cells within 30 days from 1 XI 0^s of hESC, whereas other method can only produce up to 1 x 10^s MSG cells within 30 days. it is a further object of the disclosed method to provide MSCs having high immunosuppressive efficacy. The T-MSC have higher immunosuppressive potency than MSCs derived from bone marrow (B ) or other sources, the T-MSC have higher immunosuppressive potency than MSCs derived from hESCs via other methods.

In specific embodiments, the T-MSC suppress CD4^f or ΟΌ ^" T cell proliferation by at feast 50%, 70%, 90%, or 95% in an MLR compared to an amount of T cell proliferation in the MLR in the absence of the T-MSC.

in another specific embodiment, any of the foregoing compositions comprises a matrix, in a more specific embodiment, the matrix is a three-dimensional scaffold. In another more specific embodiment, the matrix comprises collagen, gelatin, laminin, ffbronectin, pectin, ornithine, or vitronectin, in another more specific embodiment, the matrix is a biomaterial. in another more specific embodiment, the matrix comprises an extracellular membrane protein, in another more specific embodiment, the matrix comprises a synthetic compound. In another more specific embodiment, the matrix comprises a bioactive compound. In another more specific embodiment, the bioactive compound is a growth factor, cytokine, antibody, or organic molecule of less than 5,000 da!tons.

The invention further provides cryopreserved stem cell populations, e.g., a cell population comprising T-MSC, wherein the cell population is immunomodulatory, which are described herein. For example, the invention provides a population of T-MSC that have been identified as detectabiy suppressing T cell proliferation and/or differentiation in a mixed lymphocyte reaction {MLR) assay, wherein the cells have been cryopreserved, and wherein the population is contained within a container.

In a specific embodiment of any of the foregoing cryopreserved populations, the container is a bag. In various specific embodiments, the popuSation comprises about, at least, or at most 1 x 10* the stem ceils, 5 x 10⁶ the stem ceiis, 1 x 10⁷ the stem celis, 5 x 10⁷ the stem ceiis, 1 x 10³ the stem celis, 5 x 10⁸ the stem ceils, 1 x 10⁹ the stem ceils, 5 x1 0^s the stem celis, or 1 x 1 G^,G the stem cells, in other specific embodiments of any of the foregoing cryopreserved populations, the stem ceils have been passaged about, at least, or no more than 5 times, no more than 0 times, no more than 15 times, or no more than 20 times, in another specific embodiment of any of the foregoing cryopreserved populations, the stem ceiis have been expanded within the container.

4, BRIEF DESCRIPTION OF FIGURES

FIGS 1 (A-B). (A) Flow chart of the protocol for hESC differentiation into T-MSCs via a trophobiast and pre-T-MSC stage. Key bio-markers that are associated with each differentiation stage are indicated. (B) Comparison of various MSG generation protocols for MSC yield and quality: hESCs were differentiated in three protocols. 1 ) T-MSC; 3 days in the trophobiast differentiation medium followed by 8-10 days in a MSC growth medium, 2} SB-MSC: 3-10 days in SB431542-suppiemented differentiation medium followed by 12 days in the MSC growth medium, 3) H8-MSC; hESC are differentiated into MSC throug a hemangiob!ast intermediate stage, hESC wer differentiated into hemangioblast in serum-free medium for 10-13 days followed by 12 days in the MSC growth medium. The tota! number of MSCs (millions of cells) in different cultures at day 10, 20 and 30 following the initiation of the differentiation procedures are shown. MSG purity was determined by FACS analysis of CD73+ cell ratio, .

FiGS 2 { A-C), Morphological changes observed at various time points in cultures of hESCs which are in the process of differentiating to T-MSCs. (A) Day 2: trophobiasts; (B) Day 5: pre-MSCs {mesodermal cells}; and {C} Day 9: MSCs.

F!GS 3 (A-C), Analysis of the ratio of cells expressing the trophobiast marker Trop-2 (Trp-2) and MSC marker CD73 at various time points during the differentiation of hESC into T- MSC. (A) Day 2: trophobiasts; (B) Day 5: pre-MSCs (mesodermal ceiis); and (C) Day 9: MSCs.

FIGS 4 (A-H). Surface marker expression profile of T-MSC after 1 1 days of differentiation, (A) Trp2 is a marker for trophobiasts, (B) CD31 is a marker for endothelial cells, and (C) CD34 is a marker for hematopoietic stem cells. (D-H) CD73, CD90, CD 105, CD44, CD29 are markers for MSCs.

FIGS 5 (A-R). The in vitro immunosuppressive function of T-MSCs. BM-MSCs (G-L) or T-

MSCs (M-R) were mixed with CFSE-iabeled mouse lymphocytes at 10:1 ratio. The cells were stimulated with anti-CD3 antibody at 0.3 or 1 pg/ml together with 1 pg/rnl of anti-CD28 antibody. Ceil proliferation was indicated by CFSE dilution via FACS anaiysis. (A-F) T cells cultured without 8M-MSC or T-MSC (labeled control) are shown,

FIG 6 . T-MSC attenuate the disease score of an EAE mouse model; EAE was induced in G5781/6 mice with MOG35-55 plus an adjuvant and pertussis toxin, T-MSC, BM-MSC or MSCs derived from hESCs using the SB431542 method (hES-MSC(SB)} were intraperitoneously injected into the mice, 6 days after the EAE induction. Disease score {from 0 being the no disease to 4 being the severe disease) was recorded for 27 days after the MSG injection.

FIGS 7 (A-C). Determination of the muStipoiency of T-MSC to differentiate into: (A) osteocytes, (8) chondrocytes, and (C) adipocytes.

FIG 8, Gene expression anaiysis of comparing hES-HB- SC (hES hemagiobiast derived MSC) with T-MSC (hES trophob!ast derived MSG) and 8M- SC {aduit bone marrow derived MSG). Gene expression was normalized and is shown as arbitrary expression units.

5, DETAILED DESCRIPTION

5.1 DEFINmONS

The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provid additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated thai the same thing can be the in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein , nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments.

The term hESC means human embryonic stem cells that encompass piuripotent stem celis produced from embryo, inner cell mass, b!astamere or a cell line,

The term "hES-MSC" or hES-MSCs" or "human embryonic mesenchymal stem cells" or human embryonic stem eel! derived mesenchymal stem cells" or "hES-MSC population" as used herein means mesenchymal-like stem ceils_; mesenchymai-iike stromal cells, mesenchymal stem cells or mesenchymal stromal cells, derived from human embryonic stem celis or derived from induced piuripotent stem ceils ("iPSCs-^'} using any methods. hES-MSC as used herein includes individual cells, cell Sines, batches, lots or populations of hES-MSC.

The term "T-MSC" refers to MSC or mesenchymal stem/stromal celis that are derived from human embryonic stem ceils (hESC) or induced piuripotent stem celis (iPSC) through a tropboblast intermediate stage where cells express Trop-2 with trophoblast-iike morpho!ogy. The term "hES-T-MSC" refers to T-MSC differentiated from hESC. The term "iPS-T-MSC" and 1T- MSG" refer to T-MSC differentiated from iPSC. The term "T-MSC" as used herein does not refer to a trophob!ast. A cell is considered a "stem ceil" if the cell retains at least one attribute of a stem cell, e.g., the ability to differentiate into at least one other type of cell, or the like. These ceils can be described based upon numerous structural and functional properties including but not limited to, expression or lack of expression of one or more markers. T-MSCs_; including both hES-T-MSC and iT- SC, are multipotent and capable of differentiating to give rise to other cell types and ceil lineages.

The term "hES-H8~MSC^s and "HB-MSC" are mesenchymal stem cells that are derived from human piuripotent stem cells including hESC and iPSCs via hemangiob!ast or hemangio- colony forming middle step.

The term "clinical grade T-MSC" as used herein means T-MSC which contains characteristics that are suitable for use in clinical use fo human, avian or other mammals. Clinical grade T-MSC as used herein includes individual cells, ceil lines, batches, lots or populations of MSG.

The term "T-MSC population" as used herein means a population of T-MSC cells which contains ceils that have characteristics that are suitable for use in treatment and cells that do not have characteristics that are suitable for use in treatment,

The term -Fv SC derived lineages'¹ or T-MSC-DL as used herein means ceils or cell lineages differentiated from T-MSC including, but not limited to, adipocytes, myoblast ceils, neural lineage ceils, osteoblast cells, fibroblast, chondrocytes, and stromal cells.

The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to cause an improvement in a clinically significant condition in the subject, or deiays or minimizes or mitigates one or more symptoms associated with the disease, or results in a desired beneficial change of physiology in the subject.

The terms "treat", "treatment", and the like refer to a means to slow down, relieve, ameliorate or alleviate at least one of the symptoms of the disease, or reverse the disease after its onset.

The terms "prevent", "prevention", and the like refer to acting prior to overt disease onset, to prevent the disease from developing or minimize the extent of the disease or slow its course of development

The term "subject" as used in this application means an animal with an immune system such as avians and mammals. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates. Avians include, but are not limited to, fowls, songbirds, and raptors. Thus, the invention can be used in veterinary medicine, e.g. , to treat companion animate, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical applications

The term "in need thereof would be a subject known or suspected of having or being at risk of developing a disease including but not limited to multiple sclerosis and other T cell related autoimmune diseases, or diseases related to the central nervous system or the blood-brain barrier or the blood-spinal cord barrier.

A subject in need of treatment would be one that has already developed the disease. A subject in need of prevention would be one with risk factors of the disease.

The term "agent" as used herein means a substance that produces or is capable of producing an effect and would include, but is not limited to, chemicals, pharmaceuticals, drugs, biologies, small molecules, antibodies, nucleic acids, peptides, and proteins.

As used herein, a stem cell is "positive" for a particular marker when that marker is detectable. For example, a T- SC is positive for, e.g., CD73 because CD73 is detectable on T- MSC in an amount detectab!y greater than background (in comparison to, e.g., an isotype control}. A cell is also positive for a marker when that marker can be used to distinguish the cell from at least one other cell type, or can be used to select or isolate the cell when present or expressed by the cell.

As used herein, "immunomodulation" and "immunomodulatory" mean causing, or having the capacity to cause, a detectable change in an immune response, and the ability to cause a detectable change in an immune response.

As used herein, "immunosuppression" and "immunosuppressive" mean causing, or having the capacity to cause, a detectable reduction in an immune response, and the ability to cause a detectable suppression of an immune response.

The present invention is based on the first discovery that mesenchymal stem cells MSCs can be differentiated from the hESC derived trophoblasfs, and that the trophoblast-derived MSCs (T-MSC) can be used for tissue repair and immune regulation. These T-MSC produced from the disclosed methods all remarkably inhibited T cell proliferation and differentiation in viitx> and attenuated the disease score in vivo, whereas bone marrow-derived MSG (BSV1-MSC) had no effect at all in vivo, although the BM-MSC may partially reduce T ceil proliferation and differentiation in vitro. The T-MSC disclosed herein have surprisingly higher immunosuppressive activity compared to BM-MSC. The methods disclosed herein are highly efficient and can produce high number of T-MSC with low cost and high purity. The methods disclosed herein are highly reproducible with little batch-to-batch variations, and easily adaptable to meet clinical needs.

Thus, the present invention overcomes the problems described above by providing a method of generating mesenchymal stem eels (MSG) in vitro from human embryonic stem cells. The ability to generate the hES-T-MSC by the methods disclosed herein allows the production of cells that can foe used in a variety of therapeutic applications, including the treatment and prevention of multiple sclerosis, and other autoimmune diseases. Additionally, the hES- SC produced by the methods described herein have the ability to cross the brain-blood barrier (BBB) and the blood-spinal cord barrier (BSCB) allowing them to be used for a variety of therapeutic applications, including drug delivery. The methods of the invention provide further utility in that they enabie the generation of large numbers of hES-T-MSC that can be used on a commercial scale.

5.2 Differentiation of Embryonic Stem Cells through TrophobSast to Obtain T-IVISC Disclosed herein is a method for generating and expanding mesenchymal-Sike stem cells

( SCs) from trophoblast derived from embryonic stem cells (hES), These resulting cells are designated T-fVlSC, These T-fVlSC can be isolated and/or purified.

MSC-iike ceils have been derived from human embryonic stem ce!is by various methods (Barbieri et a/. (2005); Olivier ei at. (2006): Sanchez et a/. {2011 }: Brown et a/. (2009)). However, a!i of these methods involve co-euituring and hand-picking procedures that limit yield and purity and result in varying quality of cells.

Although hESC express low levels of MHC antigens, it has been found that many cell types differentiated from hESC have increased expression of these antigens (Draper et a/., 2002; Drukker ef a/., 2006; Drukker el a/., 2002), thus, causing great concern for irnmunorejeefion of the differentiated cells if transplanted into patients. In contrast, MSG express low levels of costimulatory molecules and major MHC antigens, and have been used in allogeneic or xenograft models to treat autoimmune diseases (Gordon et at., 2008b; Grinnemo ef a/,, 2004; Rafei ef a/.,2009a; Rafei et at., 2009b; Tse ei a/., 2003). T- SC, like adult tissue-derived MSC, express Sow levels of the co~sfirnulatory molecules and MHC antigens, and do not require long- term engraftment to exert immunosuppressive effect, thus, there is no concern for immunorejection due to mismatch of MHC antigens between SC and the recipient. One hESC line is sufficient to generate T-MSC at Iarge scale, in an endless supply, and with easy quality control, suitable for industrial production as a potential therapy to treat patients with MS and other T cell-based autoimmune diseases.

Human trophoblast can be generated from human embryonic stem cells. Such embryonic stem cells include embryonic stem cells derived from or using, for exampie, blastocysts, plated ICfvls, one or more blastomeres, or other portions of a pre-impSantafion-stage embryo or embryo- like structure, regardless of whether produced by fertilization, somatic ceil nuclear transfer (SCNT), parthenogenesis, androgenesis, or other sexual or asexual means.

Additionally or alternatively, trophobiast can be generated from other embryo-derived cells. For exampie, trophoblast can be generated (without necessarily going through a step of embryonic stem cell derivation) from or using plated embryos, !CrVls, blastocysts, one or more biastomeres, trophoblast stem eels, embryonic germ cells, or other portions of a pre- implantation-stage embryo or embryo-like structure, regardless of whether produced by fertilization, somatic cell nuciear transfer (SCNT), parthenogenesis, androgenesis, or other sexual or asexual means. Similarly, trophoblast can be generated using cells or cell lines partiaiiy differentiated from embryo-derived cells. For example, if a human embryonic stem ceil line is used to produce ceils that are more developmental primitive than trophoblast, in terms of development potential and plasticity, such embryo-derived ceils could then be used to generate trophobiasi.

Additionaiiy or alternatively, trophoblast can be generated from other pre-natal or peri- nata! sources including, without limitation, umbilical cord, umbilical cord blood, amniotic fluid, amniotic stem ceils, and placenta.

The human embryonic stem cells may be the starting material of this method. The embryonic stem cells may be cultured in any way known in the art, such as in the presence or absence of feeder ceiis.

In the examples set forth herein, eight hESC ceil !ines were used, H9 (derived from

WiCei! Research Institute) (Thomson ef a/. (1998), CT2 (derived from University of Connecticut Stem Ceil Core (Lin et a/. (2010)); and ES03~Envy (Envy, a GFP-labeied line, derived at ES International) (Costa et at. (2005}), ESI-017, ESI-053, ESI-049, ESI-035, and ESS-Q51.

In the first step of this method to obtain T- SC, human embryonic stem celis are grown in small e!urnps or single ceiis in serum-free media without bFGF. The celis are then re-plated and cultured with 8MP4 (1-200 ng/mi ) as the only cytokine for a short time (2-5 days) to obtain a highly homogenous population of trophobSasts as they express the typical trophoblast marker Trop2/TACSTD2 (Trp2). A TGFp inhibitor (SB431542 (1-20μΜ), A83-01 (0.2-5 μΜ) or ALK5 inhibitor (1-20 μΜ), etc.) can be used to increase the trophoblast forming efficiency. The cells will expand and differentiate into trophoblast celis in 2-5 days with irophobSast-like morphology, in certain embodiments, more than 90% of ceiis express Trop-2/TACSTD2 (Trp-2) (Xu et a!,, 2002). Trophoblasts may be isolated by size or purified with antibody, such as by immunoaffinity eo!umn chromatography.

In one embodiment, trophoblast celis are digested to form single cells with TryplE, Trypsin or coiiagenase B. The sing!e cells are re-suspended in a medium optimized for mesenehymai stem eel! growth such as alpha-MEM containing 2-20% of fetal bovine serum (FBS) o human AB serum (ABHS), DME -high glucose containing 2-20% of F8S or A8HS, the FBS can be replaced with 5-20% of knock-out serum replacement (KOSR) or bovine serum aibumin (SSA), or any other commercial available serum free MSG culture mediums, in certain embodiments, Serum, KOSR or BSA is added in a concentration of from about 5-20%. In certain embodiments, fetal bovine serum is preferred. In certain embodiments, ceils are cultured at a density of about 10-1000 ceils/cm², in certain embodiments, the ceils are cultured in an environment that mimics the extrace!iular environment of tissues, such as gelatin, vitronectin, laminin, fibronectin, collagen I. In certain embodiments, the MSG culture medium comprises LIF (2-2Gng/ml), bFGF (2-100ng/ml), or PDGF (1 -5Qng/ml) to increase expansion efficiency.

After approximately 24 hours, a number of ceils (50-90%) attached to the culture plate and approximately 2-3 days later, pre-T-MSC begin to differentiate from the irophoblasts, cells were elongated and form clear cell border, in certain embodiments, the pre-T-MSC express both CD73 and Trop-2. After 6-10 days, more than 80-90% cells irophoblasts are differentiated into mesenchymal-like small ceil with spindle-like morphology, so called T-MSC here. T-MSC can also be identified by the expression of certain markers, such as CD73, CD90, CD 105, CD13, CD29, CD54, CD44, CD146 and CD166 and by the absence or low expression of certain markers such as CD31 , CD34, and CD45. in certain embodiments, T-MSC do not express HOX and HLA-G, In certain embodiments, T-MSC express high ievei of CXCR7, CXCL2, CXCL12 but low level of HOXB2, HOXB3, HOXB5, HOXB7, HOXB9, HOXA5, HOXA9 and other HOX family genes, T-MSC are also characterized as muitipotertt and able to differentiate into adipocytes, chondrocytes, osteoblast celis, neurons, myoblasts, stroma! ceils and fibroblasts.

Provided herein is an isolated cell population comprising a plurality of immunosuppressive T-MSC thai expresses at least one of the following markers: CD73, CD90 and CD105.

In a further embodiment of the present invention, an additional step of irradiating the T~ MSCs is performed. This irradiation can be accomplished with the use of any method known in the art that emits radiation including but not limited to gamma irradiation e.g., Cesium-137 gamma irradiation, or photon radiation using X-ray. The preferred amount of radiation to be administered is about between 5 and 20000 gy, more preferably about between 50 and 100 gy, and most preferably 80 gy.

In one embodiment, the method described herein is a novel process for deriving {also referred to herein as producing) T-MSC from hESCs. Th method comprises the steps of;

a. Culturing a cell culture comprising human embryonic stem cells in serum- free medium in the present of at !east one growth factor in an amount sufficient to induce the differentiation of the embryonic stem celis to differentiate into trophobiast; in an embodiment, the time period of the differentiation into trophobiast is about 2-5 days; in an embodiment, the medium comprises BMP4, with or without the presence of an TGFb inhibitor (i.e., SB431542, A83-01 or ALK5 inhibitor etc.) to increase the differentiation efficiency;

b. Adding at least one growth factor to the culture comprising the trophobiasts and continuing to cuiture in serum-free medium, wherein the growth factor is in an amount sufficient to expand the trophobiasts, in an embodiment, the medium comprises B P4, (this step is optional); c. isolating the trophobiasts and re-piating the trophobiasts onto gelatin, Iaminin, fibronectin, vitronectin, collagen or Mairigei-coated plates and cultured in a serum-containing or serum-free media in an amount sufficient to differentiate the trophoblast into ΊΓ- SC through pre-T-MSC, in an embodiment, the isolated trophoblast is cultured for 6-10 days to produce the T-MSC, wherein at least about 90%, 95%, 96%,

97%, 98%, 99% of the resulting T-MSC express ceil surface markers for adult MSCs, in an embodiment, the medium comprises LiF, bFGF, PDGF to increase expansion efficiency,

wherein at least about 90%, 95%, 96%, 97%, 98%, 99% of the resulting T-MSC express ceil surface markers for adult MSCs.

As shown in Figs, 1 & 2, the disclosed method starts with dispersal of hESC colonies into small clumps or single cells. The eels are then re-plated and cultured with BMP4 as the only cytokine, and a TGF inhibitor for a short time (2-5 days) to obtain a highly homogenous population of trophobiasts as they express the typical trophoblast marker Trop-2/TACSTD2 {Trp- 2} (Xu et ai. , 2002). The trophobiasts are then dissociated and re-plated onto a gelatin, Iaminin, fibronectin, vitronectin, collagen or matrigel-coated plate and cultured in a IV SC growth medium fo 4-10 days to generate spindle-like ceils similar to the morphology of typical MSCs.

The method disclosed herein, uniike the other methods, does not requir feeder cells, sorting or hand-picking of the cells. The initial trophoblast differentiation step is in a defined, serum-free medium without bFGF. The entire protocol only requires two steps of differentiation in a total of 6-14 days to generate T-MSC at high purity and high yie!d (Fig. 1 ). This is the shortest differentiation protocol ever reported for MSC derivation from hESC. The yield and purity of the T-MSC are very high compared to those achieved using previously reported methods. Within 30 days, T-MSC at 5 x 10^s fold the number of the original hESCs can be obtained and with a high percentage of CD73+ cells, a typical marker for MSCs, whereas the other methods can only yield less than 100 fold the original hESC number with a iow percentag of CD73+ cells. Th derivation of the T-MSC includes an intermediat stag of CD73 Trp-2 double positive celis, hereafter named pre-T-MSC. After 2-3 days of the BMP4 plus a TGFp inhibitor treatment, the celis first express Trp-2 at a high percentage and demonstrate a homogenous morphology of trophobiasts (Figs. 2 & 3). After 5-6 days, the ceils express both Trp-2 and CD73; after 6-14 days, the ce!is no longe express Trp2 but express the typicai MSG surface markers at hig percentages including CD73 (>98%), CD90 (>95%), CD105 (>90%), CD44 (>95%), CD29 (>80%); and the cells are negative for the endothelial marker CD31 and hematopoiesis markers CD34 and CD45 (Figs. 3 & 4).

T-MSC produced by the method disclosed herein are capable of differentiating to downstream osteogenesis, chondrogenesis and adipogenesis iineages (Fig. 7). Thus, the T-MSC are phenotypically and functionally similar to SCs derived from the bone marrow (BM) and other sources.

5.3 Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells Bone marrow-derived MSCs (BM-MSCs) have long been used to treat autoimmune disease in many animal modeis and clinical trials, however the efficacy of immunosuppression is not consistent with some reports showing BM-MSCs are unable to efficiently treat certain autoimmune diseases (Tynda!i, 2011 ), Data is provided herein comparing the ability of BM-MSCs and T-MSC for their inhibition of T cell proliferation following T cell receptor stimulation. As shown in Fig. 7, BM-MSCs can inhibit proliferation of both CD4 and CD8 T cells induced by anfi- CD3 antibody at a low dose {0.3 ug/ml), which is comparable to T-MSC. However, when the anti- CD3 antibody concentration increased to 1ug /ml, BM-MSCs have less potency in suppressing proliferation of both CD4 and CDS T cells than T-MSC. CFSE dilution assay was used here to evaluate the T cell proliferation: an increased percentage of T cells with decreased CFSE signal indicates an accelerated proliferation. As shown in Fig. 5, when anti-CD3 antibody increased to 1 ug/mi, there were 59% of CD4 and 48% of CD8 T ceils detected with decreased CFSE signal, T- MSC significantly decreased both the CD4 and CD8 T cells to 16%, whereas BM-MSCs only decreased CD4 and CDS T cells to 32% and 36%, respectiveiy.

Consistent with the in vitro immunosuppressive activity of the T-MSC, T-MSC produced by the method disclosed herein were shown to be effective to treat experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. As shown in Fig. 6, when T-MSC were injected 6 days post the EAE induction, the disease score of the EAE mice significantly declined, compared to vehicle injection controls.

In a further feature of cells produced by the disclosed methods, T-MSC also demonstrated much stronger immunosuppressive effect than BM-MSCs and hES-MSCs derived through SB431542 treatment (Chen et ai., 2012) {Fig. 6), in several repeated experiments, BM- MSCs consistently failed to attenuate the disease score of EAE mice. Thus, the replacement of BM-MSCs with T-MSC produced by the disclosed method for use in clinical applications wouid remove the need for risky, invasive procedures for bone marrow aspiration, reduce the time for waiting for BM donations, reduce the cost, and reduce batch to batch variations for preparing BM-MSCs on a per-pa ient basis.

in summary, disciosed herein is a highly efficient method to generate mesenchymal-like cells or MSCs from hESCs throug an intermediate trophobiast stage, and the use of the T-MSC to treat autoimmune disease. Microarray anaiysis suggested that the T-MSC had a gene expression profile not identical to that of BM-MSCs (data not shown), although both can differentiate into the same downstream cell lineages (Fig, 7). !n addition, the T-MSC have stronge immunosuppressive ability both in vitro and in vivo than BM-MSCs. The available data suggest that T-MSC produced by the disclosed method are different from traditional, aduit-derived SCs. Due fo their strong inhibition of T cell proliferation, T-MSC may be used to treat multiple sclerosis with much higher efficacy than BM-MSCs. To address potential safety concerns, T-MSC were injected into immunodeftcieni SClD-beige mice. No tumor or teratoma formation was observed in the mice.

The T-MSC of the present invention are unique and have a variety of therapeutic and other uses. Thus, the present invention includes various preparations, including pharmaceutical preparations, and compositions comprising T-MSC,

The term "T-MSC" refers to MSG or mesenchimal stem/stromal ceils that are derived from human embryonic stem ceils (hESC) or induced plurspoteni stem cells (iPSC) through a trophob!ast intermediate stage where ceils express Trop-2 with frophobiast-lke morphology. The term "hES-T-MSC" refers to T-ivlSC differentiated from hESC. The term "iPS-T-MSC" and ΊΤ- MSC" refer to T-MSC differentiated from iPSC. The term "T-MSC" as used herein does not refer to a trophobiast. A eel! is considered a "stem cell" if the ceii retains at least one attribute of a stem ceii, e.g. , the ability to differentiate into at least one othe type of ceii, or the like. These cei!s can be described based upon numerous structural and functional properties including but not limited to, expression or lack of expression of one or more markers. Specifically, T-MSC are characterized by small cell bodies with a fibroblast morphology. T-MSCs, including both hES-T- MSC and ίΤ-MSC, are muitipotent and capable of differentiating to give rise to other ceii types and ceil lineages. The term "T-SvlSC-DL" refers to all the cell types and cell lineages differentiated from T-MSC,

The differentiation method described herein can achieve the differentiation of MSC from iPS ceils within 6-14 days, the shortest time ever reported. Thus, these iT~MSC can be used for patient specific iPS based therapy under emergency conditions which requires the generation of MSC in very short time, such as acute heart infarction, acute heart failure, acute spinal cord injury, acute radiation/burning treatments, etc.

T-MSC can be identified or characterized by the expression or lack of expression as assessed on the level of DNA, RNA or protein, of one or more ceil markers, T-MSC can be identified as expressing ceil surface marker CD73, or expressing at least one or more of the following cell surface markers: CD90, CD105, CD13, CD29, CD54, CD44, CD146 or CD166 or not expressing or expressing at a low level at least one of the following ceil surface markers^': CD34, CD31 , or CD45.

Alternatively or additionally, T-MSC can be identified or characterized based upon their low level of expression of one or more pro-inflammatory proteins, MMP2, RAGE, IFNGR2, TNFa, IL-12A, 11-6, and VCAM1. This profile of gene expression is in contrast to bone marrow derived mesenchymal stem ceils. In particular, IL-6 was expressed much higher in BM-MSCs than in T- MSG. fL-6 is a pieiotropie cytokine involved in crosstalk between hematopoietic / immune ceils and stromal cells, including the onset and resolution of inflammation.

The T-MSC can also be characterized in their ability to inhibit T ceii proliferation after stimulation in vitro. This characteristic is in contrast to BM-MSGs which do not inhibit T ceii proliferation after simulation in vitro.

Thus, the T-MSC described herein have at least one of the following characteristics: (1 ) differentiate into adipocytes, chondrocytes, osteoblast cells, neurons, myoblasts, stromal cells and fibroblasts; (2) have a fifarobiast-iike morphology; (3) express CD73, CD90, CD 105, CD13, CD29, CD54, CD44, CD 146 and/or CD166; (4) express at low ieveis or do not express CD34, CD31 , and /or CD45; {5} express at low ieveis or do not express M P2, RAGE, IFNyRi IFNyR2, IL-12, TNFcs, IL-6, and/or VCAM1 , particularly !L~6; (6) express IV5HC antigen HLA-G and/or HLA-ABC and express at low levels or do not express HLA-DR and/or CD80; and (7) inhibit T celi proliferation after stimulation in vitro, in certain embodiments, the T-MSCs have at least two, at least three, at least four, at least five, at least six, or aii seven characteristics,

In certain embodiments, T-MSC is distinguishable with previously reported HB-IV5SC, T-

MSC express at least one fold higher !eve! of CXCR7, CXCL2 and/or CXCL12 than HB-MSC, but at ieast half of the level of HOXB2, HOXB3, HOXB5, HOXB7, HOXB9, HOXA5, HOXA9 and other HOX family genes compared to HB-MSC.

Additionaiiy, the T-MSC have the unique abiiity to cross the blood-brain barrier (BBB) and the blood-spinal cord barrier {BSCB}, making them uniquely suited for therapeutic and diagnostic applications. The T-MSC of the current invention have the abiiity to migrate in and out of the vessels of the spinal cord, across the BSCB, to fulfill functions in the CNS, including but not iimited to the delivery of therapeutic and diagnostic agents. This is in contrast to BM-MSCs which do not have this ability.

Another embodiment of the present invention is a T-MSC that is irradiated. This embodiment would include T-MSC with at ieast one of the following characteristics listed above, having at least two, at ieast three, at least four, at least five, at ieast six, or ail seven characteristics that have been subject to irradiation.

in another embodiment, the ceil culture comprises T-MSC. in certain embodiments, the T-MSC differentiate into adipocytes, chondrocytes, osteoblast cells, neurons, myoblasts, stroma! cells and fibrobiasfs. in certain embodiments, the T-MSC cells express CD73, CD90, CD105, CD13, CD29, CD54, CD44, CD146, and/or CD166. in certain embodiments, the ceils express at low ieveis or do not express CD34, CD31 , and/or CD45. In certain other embodiments, the cells express at Sow ieveis or do not express MMP2, RAGE, IFNyRi , !FNvR2, IL-12, TNFa, IL-6, and/or VCAM1 , especially IL-6. !n certain other embodiments, the cells express MHC antigen HLA-G and/or HLA-ABC and express at low levels or do not express HLA-DR and/or CD80. in certain other embodiments, the cells inhibit T cell proiiferation after stimulation in vitro. In certain embodiments, the eels can cross the biood-brain barrier and the blood-spinal cord barrier. In certain embodiments, the cells have been irradiated.

In another aspect, disclosed herein a pharmaceutical preparation comprising T-MSC, In certain embodiments, the T-MSC can differentiate into adipocytes, chondrocytes, osteobSast cells, neurons, myoblasts, stromal cells and fibroblasts. In certain embodiments, the cells express CD73, CD90, CD105, CD13, CD29, CD54, CD44, CD146 and/or CD166. In certain embodiments, the cells express at low levels or do not express CD34, CD31 , and/or CD45. In certain other embodiments, the cells express at Sow levels or do not express MMP2, RAGE. IFNyRI , iFNyR2, TNFct, SL-12, SL-6, and/or VCAM1 , especially IL-6. Sn certain othe embodiments, the ceils express MHC antigen HLA-G and/or HLA-ABC and express at low levels or do not express HLA-DR and/or CD80. in certain other embodiments, the ceils inhibit T cell proliferation after stimulation in vitro. In certain embodiments, the cells can cross the biood-brain barrier and the blood-spinal cord barrier. In certain embodiments, the cells have been irradiated. The pharmaceutical preparation can be prepared using any pharmaceutically acceptable carrier or excipient.

In certain embodiments, the composition or pharmaceutical preparation comprises at ieast at Ieast 10,000 T-MSC, at ieast 50,000 T-MSC, at least 100,000 T-MSC, at ieast 500,000 T-MSC, at ieast 1 x 10⁵ T-MSC, at least 5 x 10^s T-MSC, at least 1 x 10⁷ T-MSC, at Ieast 5 x 10⁷ T-MSC, at Ieast 1 x 10^s T-MSC, at Ieast 5 x 10^s T-MSC, at least 1 x 10^s T-MSC, at Ieast 5 x 10^δ T-MSC, or at least 1 x 10¹⁰ T-MSC.

Provided herein are pluralities of T-MSC that comprise T-MSC obtained and isolated directly from a human embryonic stem cell line that have been cultured and passaged at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30 or more times, or a combination thereof.

In certain embodiments, provided herein is a cryopreserved preparation of T-MSC or ceils partially or terminally differentiated therefrom.

In certain embodiments, provided herein is a therapeutic use of T-MSC, or compositions or preparations of T-MSC, including irradiated T-MSC, Such ceils and preparations can b used in the treatment of any of t e conditions or diseases as described, as well as in a delivery system for agents across the biood-brain barrier and the blood-spinal cord barrier.

In certain embodiments, the invention provides a cryopreserved preparation of trophoblasts, pre-T-MSC, or T-MSC cells partially or terminally differentiated therefrom.

In certain embodiments, the invention provides the therapeutic use of T-MSCs, or compositions or preparations of T-MSCs, including irradiated T-MSCs. Such cells and preparations can be used in the treatment of any of the conditions or diseases detailed throughout the specification, as well as in a delivery system for agents across the blood-brain barrier and the b!ood-spinal cord barrier. 5.4 Selecting and Producing T-MSC Populations

Provided herein is a method of identifying highly immunosuppressive T-MSC by identifying a biomarker profile of the highly immunosuppressive T-IVISC that are clinicai grade for use in therapy. In certain embodiments, the clinical grade T-MSC have the following characteristics: ø) contain >95% of cells expressing group- markers; (ii) contain 80% of cells expressing group 2 markers; (iii) contain <5% of cells expressing group-3 markers (iv) express IL-1Q and TGFP; (v) contain <2% of cells expressing lL-6, iL-12 and TNFa; and (vi) contains <0.QQ1% of cells co-expressing ail group-4 markers, wherein group-1 markers are CD73, CD90, CD105, CD146, CD166, and CD44, group-2 markers are CD13, CD29, CD54, CD49E, group-3 markers are CD45, CD34, CD31 and SSEA4, and group-4 markers are OCT4, NANOG, TRA-1- 60 and SSEA4.

In certain embodiments, the method comprises measuring the differential expression of markers that encode anti-inflammatory factors ("AIF") and pro-inflammatory factors fP!F"}. in certain embodiments, the AIF is IL-10, TGF£2. in certain embodiments, the PIF is up regulated. In certain embodiments, T-MSC express at least 1.5 fold of the above markers as compared to BM-MSC. In certain embodiments, the PIF is IL-6, iL-12, TNFa, CCL2, VCAM1 , RAGE, MMP2, In certain embodiments, tie P!F is down regulated. In certain embodiments, T-MSC express at ieast half of the above markers as compared to BM-MSC in another embodiment, highly immunosuppressive T-MSC has a iower ratio of IL-6* cells as compared to BM-MSC. in certain embodiments, highly immunosuppressive T-MSC have less than 5%, 4%, 3%, 2%, or i % of IL-6 positive celis. In certain embodiments, T-MSC express low SeveSs of 1L12, TNFa, RAGE and other PIF. in certain embodiments, T-MSC may express high levels of TGFp2 and SL-10. In certain embodiments, the expression of markers is compared to expression in BM-MSC.

Provided herein is a qualification procedure for clinicai grade T-MSC population. Expression of specific markers is measured in a population of T-MSC to determine whether they are suitabie for therapeutic use. The markers inciude, for example, {1 ) MSC-specific markers (set 1 ): CD73, CD90, CD105, CD166, and CD44, (2) MSC-specific markers (set 2): CD13, CD29, CD54, CD49E, SCA-1 , and STRO-1 , (3) hematopoietic stem/progenitor markers; CD45 and CD34, and endothelial celi marker CD31 , (4) immunogenic markers; HLA-ABC, HLA-G, CD80, and CD86, (5) cytokines: iL-10, TGFp, !L-6, and IL-12, and (6) pluripotency markers: OCT4, NANOG, TRA-1 -6Q, and SSEA-4. In certain embodiments, T-MSC population contains more than 95%, 96%, 97%, 98%, or 99% of cells thai express at least one group 1 markers. In certain embodiments, T-WSC population contains more than 80%, 85%, 90%, 95%, or 99% of ceils that express at ieast one group 2 markers. In certain embodiments, T-MSC population contains less than 0.1 %, 0.08%, 0.05%, 0.03%, 0.02%, or 0.01 % of celis that express at least one group 3 marker, in certain embodiments, T-MSC population contains more than 80%, 85%, 90%, 95%, or 99% of ceils that express IL-10 and/or TGFji in certain embodiments, T-MSC population contains less than 5%, 4%, 3%, 2%, 1 % of cel!s that express !L-6 and/or IL-12. In certain embodiments, T- SC population contains iess 0,001 % of cells that express at least one group 6 marker. The c!inicai-grade T-MSC is compared with the preclinica!-grade T-MSC as a positive control. In certain embodiments, the T-MSC is characterized through multi-color fiow cytometry analyses and/or immunofluorescence, in certain embodiments, T-MSC popuiation express CCL2, CCL3, CCL4, CCL5, 11-1 , IL-2, IL-4, iL-6, !L-8, IL-10, 11-17, TNFa, TGF tFNy, GM-CSF, G-CSF, bFGF, CXCL5, VEGF, TPO or a combination thereof. In certain embodiments, the T- MSC population wi!l also be analyzed for (1 ) presence of exogenous materials such as endotoxin and residual cytokines/growth factors, and/or (2) genomic abnormalities (via karyotyping and whole-genome sequencing).

Provided herein is another qualification procedure for clinical grade T-MSC population. T~ MSG with better regeneration potential and immunosuppressive function may express a lower level of CD9, where CD9 expression level of Passage 1-2 T-MSC will be recorded as basal ievei, if after certain passages and procedures, the CD9 expression level increases by 2 fold, the cells will be stopped for passaging.

Methods for determining the expression profile of the T-MSC are known in the art, including but not limited to, flow cytometry, multiplex microarray, RT-PCT, Northern biot and Western biot. In certain embodiments, the expression profile of the MSG are determined by cytometric bead array based multiplex cytokine analysis, iuminex system based multiplex cytokine analysis, microarray RNA-seq, quantitative RT-PCR, Elispot Elisa, Eiisa cytokine array, flow cytometry luciferase reporter system, fluorescence reporter system, histology staining, and immunofluorescence staining.

5.4.1 Methods of Detecting Nucleic Acid Biomarkers in specific embodiments, biomarkers in a btomarker profile are nucleic acids. Such biomarkers and corresponding features of the btomarker profile may be generated, for example, by detecting the expression product {e.g., a polynucleotide or polypeptide) of one or more markers. In a specific embodiment, the biomarkers and corresponding features in a biomarker profile are obtained by detecting and/or analyzing one or more nucleic acids expressed from a marker disclosed herein using any method well known to those skilled in the art including, but not limited to, hybridization, microarray analysis, RT-PCR, nuclease protection assays and Northern biot analysis.

!n certain embodiments, nucleic acids detected and/or analyzed by the methods and compositions of the invention include RNA molecules such as, for example, expressed RNA molecules which include messenger RNA (mRNA) molecules, mRNA spliced variants as well as regulatory RNA, cRNA molecules (e.g., RNA molecules prepared from cDNA molecules that are transcribed in vitro) and discriminating fragments thereof. in specific embodiments, the nucleic acids are prepared in vitro from nucleic acids present in, or isolated or partially isoiated from a cell culture, whic are weli known in the art, and are described generally, e.g. , in Samforook et a!., 2001 , Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, Ν,Υ,), which is hereby incorporated by reference in its entirety.

5.4.1.1 Nucietc Acid Arrays

In certain embodiments, nucleic acid arrays are employed to generate features of biomarkers in a biomarker profile by detecting the expression of any one or more of the markers described herein, in one embodiment of the invention, a microarray such as a cDNA microarray is used to determine feature values of biomarkers in a biomarker profile. Exemplary methods for cDNA microarray analysis are described below, and in the examples.

In certain embodiments, the feature values for biomarkers in a biomarker profile are obtained by hybridizing to the array detectably iabe!ed nucleic acids representing or corresponding to the nucleic acid sequences in mRNA transcripts present in a biological sample

{e.g. , fiuorescentiy labeled cDNA synthesized from the sample) to a microarray comprising one or more probe spots.

Nucleic acid arrays, for example, microan-ays, can be made in a number of ways, of which several are described herein below. Preferably, the arrays are reproducible, allowing multiple copies of a given array to be produced and results from the microarrays compared with each other. Preferably, the arrays are made from materials that are stable under binding (e.g., nucleic acid hybridization) conditions. Those skilled in the art will know of suitable supports, substrates or carriers for hybridizing test probes to probe spots on an array, or will be able fo ascertain the same by use of routine experimentation.

Arrays, for example, microarrays, used can include one or more test probes, in some embodiments, each such test probe comprises a nucleic acid sequence that is complementary to a subsequence of R A or DNA to be detected. Each probe typically has a different nucleic acid sequence, and the position of each probe on the solid surface of the array is usually known or can be determined. Arrays useful in accordance with the invention can include, for example, oligonucleotide microarrays, cDNA based arrays, SNP arrays, spliced variant arrays and any other array abie to provide a qualitative, quantitative or semi-quantitative measurement of expression of a marker described herein. Some types of microarrays are addressable arrays. More specifically, some microarrays are positionally addressable arrays. In some embodiments, each probe of the array is iocated at a known, predetermined position on the solid support so that the identity (e.g., the sequence) of each probe can be determined from its position on the array (e.g., on the support or surface). In some embodiments, the arrays are ordered arrays. Mieroarrays are generally described in Draghici, 2003, Data Analysis Tools for DNA Microarrays, Chapman & Hali/CRC, which is hereby incorporated by reference in its entirety.

5.4.1.2 RT-PCR

In certain embodiments, to determine the feature values of biomarkers in a biomarker profile of !eve! of expression of one or more of the markers described herein, the feature values are measured by amplifying RNA from a sample using reverse transcription (RT) in combination with the polymerase chain reaction (PCR). In accordance with this embodiment, the reverse transcription may be quantitative or semi-quantitative. The RT-PCR methods taught herein may be used in conjunction with the microarray methods described above. For example, a bulk PCR reaction may be performed, and the PCR products may be resolved and used as probe spots on a microarray.

Total RNA, or mRNA is used as a template and a primer specific to the transcribed portion of the marker(s) is used to initiate reverse transcription. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al., 2001 , supra. Primer design can be accomplished based on known nucleotide sequences that have been published or available from any publicly available sequence database such as GenBank. For example, primers may be designed for any of the markers described herein. Further, primer design may be accomplished by utilizing commercially available software {e.g., Primer Designer 1 ,0, Scientific Software etc.). The product of the reverse transcription is subsequently used as a template for PCR,

PCR provides a method for rapidly amplifying a particular nucleic acid sequence by using multiple cycles of DNA replication catalyzed by a thermostable, DNA-dependent DNA polymerase to amplify the target sequence of interest. PCR requires the presence of a nucleic acid to be amplified, two single-stranded oligonucleotide primers flanking the sequence to be amplified, a DNA polymerase, deoxyribonucleoside triphosphates, a buffer and salts. The method of PCR is well known in the art. PCR, is performed, for example, as described in Muilis and Faloona, 1987, Methods Enzymol. 155:335, which is hereby incorporated by reference in its entirety.

PCR can be performed using template DNA or cDNA (at least 10 fg; more usefully, 1-

1000 ng) and at least 25 pmol of oligonucleotide primers. A typical reaction mixture includes: 2 μ! of DNA, 25 pmol of oligonucleotide primer, 2.5 μί of 10 PCR buffer 1 (Perkin-Elmer, Foster City, Calif.}, 0.4 μΙ of 1.25 dNTP, 0.15 μ! (or 2.5 units) of Taq DNA polymerase (Perkin Elmer, Foster City, Calif.) and deionized water to a total volume of 25 p!. Mineral oil is overlaid and the PCR is performed using a programmable thermal cycler.

Quantitative RT-PCR ("QRT-PCR"), which is quantitative in nature, can also be performed to provide a quantitative measure of marker expression levels. In QRT-PCR reverse

21 transcription and PGR can be performed in two steps, or reverse transcription combined with PGR can be performed concurrently. One of these techniques, for which there are commercially available kits such as Taqman (Perkin Elmer, Foster City, Calif.) or as provided by Applied Biosystems (Foster City, Calif.) is performed with a transcript-specific antiseose probe. This probe is specific for the PGR product (e.g. a nucleic acid fragment derived from a gene) and is prepared with a quencher and fluorescent reporter probe complexed to the 5' end of the oligonucleotide. Different fluorescent markers are attached to different reporters, a!iowing for measurement of two products in one reaction. When Taq DNA polymerase is activated, it cleaves off the fluorescent reporters of the probe bound to the template by virtue of its 5'-ίο-3' exonu ease activity. In the absence of the quenchers, the reporters now fluoresce. The color change in the reporters is proportional to the amount of each specific product and is measured by a fluorometer; therefore, the amount of each color is measured and the PGR product is quantified. The PCR reactions are performed in 96~weii plates so that samples derived from many individuals are processed and measured simultaneously. The Taqman system has the additional advantage of not requiring gel electrophoresis and allows for quantification when used with a standard curve.

A second technique useful for detecting PCR products quantitatively is to use an intercalating dye such as the commerciaiiy available QuantiTeci SYBR Green PCR (Giagen, Valencia Calif.). RT-PCR is performed using SYBR green as a fluorescent label which is incorporated into the PCR product during the PGR stage and produces a fluorescence proportional to the amount of PCR product.

Both Taqman and QuantiTeci SYBR systems can be used subsequent to reverse transcription of RNA. Reverse transcription can eithe be performed in the same reaction mixture as the PCR step (one-step protocol) or reverse transcription can be performed first prior to amplification utilizing PCR (two-step protocol). Additionally, other systems to quantitatively measure mRNA expression products are known, including Molecular Beacons®, which uses a probe having a fluorescent molecule and a quencher molecule, the probe capable of forming a hairpin structure such that when in the hairpin form, the fluorescence molecule is quenched, and when hybridized the fluorescence increases giving a quantitative measurement of gene expression.

5.4.1.3 Northern BSot Assays

Any hybridization technique known to those of skill in the art can be used to generate feature values for biomarkers in a biomarker profile. In other particular embodiments, feature values for biomarkers in a biomarker profile can be obtained by Northern blot analysis (to detect and quantify specific RNA molecules. A standard Northern blot assay can be used to ascertain an RNA transcript size, identify alternatively spliced RNA transcripts, and the relative amounts of one or more genes described herein (in particular, rriRNA) in a sample, in accordance with conventional Northern hybridization techniques known to those persons of ordinary skill in the art. In Northern blots, RNA. samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, cross-linked and hybridized wit a labeled probe. Non-isotopic or high specific activity radiolabeled probes can be used including random-primed, nick -translated, or PCR-generated DNA probes, in vitro transcribed RNA probes, and oligonucleotides. Additionally, sequences with only partial homology (e.g.. cD A from a different species or genomic DNA fragments that might contain an exon) may be used as probes. The labeled probe, e.g., a radiolabel!ed cDNA, either containing the fulMength, single stranded DNA or a fragment of that DNA sequence may be at least 20, at least 30, at least 50, or at least 100 consecutive nucleotides in length. The probe can be labeled by any of the many different methods known to those skilled in this art. The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels. These include, but are not limited to, fluorescein, rhodamine, auramine, Texas Red, ASV1C.A blue and Lucifer Yellow, The radioactive label can be detected by any of the currently available counting procedures. Non-limiting examples of isotopes include ³H, ¹ C, ³²P, ^3SS, ³⁶CI, ⁵ Cr, ⁵⁷Co, ^S8Co, ^S9Fe, ^S0Y, ^,2δΙ, ¹³ , and ^8eRe. Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric. spectrophoiometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging moiecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Any enzymes known to one of skill in the art can be utilized. Examples of such enzymes include, but are not limited to, peroxidase, beta-D-galacfosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase, U.S. Pat, Nos, 3,654,090, 3,850,752, and 4,016,043 are referred to by way of example for their disclosure of alternate labeling materia! and methods,

5,4.2 Methods of Detecting Proteins

in specific embodiments of the invention, feature values of biomarkers in a biomarker profile can be obtained by detecting proteins, for example, by detecting the expression product (e.g., a nucleic acid or protein) of one or more markers described herein, or post-translationaliy modified, or otherwise modified, or processed forms of such proteins, in a specific embodiment, a biomarker profile is generated by detecting and/or analyzing one or more proteins and/or discriminating fragments thereof expressed from a marker disclosed herein using any method known to those skilled in the art for detecting proteins including, but not limited to protein microarray analysis, immunohistochemistry and mass spectrometry. Standard techniques may be utilized for determining the amount of the protein or proteins of interest present in a cell culture. For example, standard techniques can be employed using, e.g. , immunoassays such as, for example, Western blot, immunoprecipitation followed by sodium dodecyl sulfate polyacrylarrtide gel electrophoresis, (SDS-PAGE), immunocytochernistry, and the like to determine the amount of protein or proteins of interest present in a sample. One exemplary agent for detecting a protein of interest is an antibody capable of specifically binding to a protein of interest preferabi an antibody detectab!y !abeied, either directly or indirectly.

For such detection methods, if desired a protein from the cell culture to be anaiyzed can easiiy be isolated using techniques which are vveil known to those of skill in the art. Protein isolation methods can, for example, be such as those described in Harlow and Lane, 1388, Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory Press {Cold Spring Harbor, N.Y.), which is hereby incorporated by reference in its entirety.

!n certain embodiments, methods of detection of the protein or proteins of interest involve their detection via interaction with a protein-specific antibody. For example, antibodies directed to a protein of interest. Antibodies can be generated utilizing standard techniques well known to those of skill in the art. In specific embodiments, antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or an antibody fragment (e.g., scFv, Fab or F{ab^!)₂) can, for example, be used.

For example, antibodies, or fragments of antibodies, specific for a protein of interest can be used to quantitatively or qualitatively detect the presence of a protein. This can be accomplished, for example, by immunofluorescence techniques. Antibodies (or fragments thereof) can, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of a protein of interest. In situ detection can be accomplished by removing a biological sampie (e.g., a biopsy specimen) from a patient, and applying thereto a labeled antibody that is directed to a protein of interest. The antibody (or fragment) is preferably applied by overlaying the antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presenc of the protein of interest, but also its distribution, in a particular sampie. A wide variety of well-known histological methods {such as staining procedures) can be utilized to achieve such in situ detection.

immunoassays for a protein of interest typically comprise incubating a sampie of a detectabiy labeled antibody capabie of identifying a protein of interest, and defecting the bound antibody by any of a number of techniques well-known in the art. As discussed in more detail, below, the term "labeled" can refer to direct labeling of the antibody via, e.g., coupling (i.e., physically Sinking) a detectable substance to the antibody, and can also refer to indirect labeling of the antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fiuorescently labeled secondary antibody. The sample can be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other sold support which is capable of immobilizing eels, cell particles or soluble proteins. The support cart then he washed with suitable buffers followed by treatment with the detectabiy labeled fingerprint gene-specific antibody. The solid phase support can then h washed with the buffer a second time to remove unbound antibody. The amount of bound iabe! on solid support can then be detected by conventional methods.

By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Weil-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, poiyacrylamides and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration can be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the externa! surface of a rod. Alternatively, the surface can be flat such as a sheet, test strip, etc. Preferred supports include polystyrene heads. Those skiiled in the art wiil know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

One of the ways in which an antibody specific for a protein of interest can be detectabiy labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) (Voller, 1978, "The Enzyme Linked Immunosorbent Assay {ELISA)", Diagnostic Horizons 2:1 -7, Microbiological Associates Quarterly Publication, alkersville, !Vld.; Voiler et al., 1978, J. Clin. Pathol. 31 :507-520; Butter, J. E„ 1981 , Meth. Enzymoi. 73:482-523; !V!aggio (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; ishikawa et al., (eds.), 1981 , Enzyme Immunoassay, Kgaku Shoin, Tokyo, each of which is hereby incorporated by reference in its entirety). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by specirophotometric, fluorimetric or by visual means. Enzymes which can be used to detectabiy label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-sieroid isomerase, yeast alcohol dehydrogenase, alpha-giycerophosphate, dehydrogenase, those phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, befa- ga!actosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards. Detection can also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect a protein of interest through the use of a radioimmunoassay (R!A) (see, for example, eintraub, 1986, Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrin Society, which is hereby incorporated by reference in its entirety). The radioactive isotope (e.g., ¹³¹1, ^3SS or ³H) can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

It is also possible to Sabei the antibody with a fluorescent compound. When the f!uorescentiy labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, aSiophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanide series. These metais can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethyienedtaminetetraaeettc acid (EDTA).

The antibody aiso can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemifurninescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are lumino!, isoluminol. theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioiuminescent compound can be used to label the antibody of the present invention. Bioluminescence is a type of chemiSuminescence found in biological systems in. which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioiuminescent protein is determined by detecting the presence of luminescence, important bioiuminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

In another embodiment, specific binding molecules other than antibodies, such as aptamers, may be used to bind the biomarkers. In yet another embodiment, the biomarker profile may comprise a measurable aspect of an infectious agent (e.g., Sipopolysaccharides or virai proteins) or a component thereof.

!n some embodiments, a protein chi assay (e.g. , The ProteinChip® Biomarker System, Ciphergen, Fremont, Calif.) is used to measur feature values for the biomarkers in the biomarker profile. See aiso, for example, Lin, 2004, Modern Pathology, 1 -9; Li, 2004, Journal of Urology 171 , 1782-1787; Wadsworth, 2004, Clinical Cancer Research, 10, 1625-1632; Prieio, 2003, Journal of Liquid Chromatography & Related Technologies 26, 2315-2328; Coombes, 2003, Clinical Chemistry 49, 1615-1623; Mian, 2003, Proieomics 3, 1725-1737; Lehre et at, 2003, BJU International 92, 223-225; and Diamond, 2003, Journal of the American Society for Mass Spectrometry 14, 780-765, each of which is hereby incorporated by reference in its entirety.

In some embodiments, a bead assay is used to measure feature values for the biomarkers in the biomarker profile. One such bead assay is the Sector? Dickinson Cytometric Bead Array (CBA). CBA employs a series of particles with discrete fluorescence intensities to simultaneously detect multiple soiubie anaiytes. CBA is combined with flow cytometry to create a multiplexed assay. The Becfon Dickinson CBA system, as embodied for example in the Becton Dickinson Human inflammation Kit, uses the sensitivity of amplified fluorescence detection by flow cytometry measure soiubie anaiytes in a particle-based immunoassay. Each bead in a CBA provides a capture surface for a specific protein and is analogous to an individually coated well in an ELISA plate. The BD CBA capture bead mixture is in suspension to allow for the detection of multiple anaiytes in a small volume sample.

In some embodiments, the multiplex analysis method described in U.S. Pat No. 5,981 ,180 ("the Ί80 patent"), hereby incorporated by reference in its entirety, and in particular for its teachings of the genera! methodology, bead technology, system hardware and antibody detection, is used to measure feature values for the biomarkers in a biomarker profile. For this analysis, a matrix of microparticies is synthesized, where the matrix consists of different sets of microparticies. Each set of microparticies can have thousands of molecules of a distinct antibody capture reagent immobilized on the microparticSe surface and can be color-coded fay incorporation of varying amounts of two fluorescent dyes. The ratio of the two fluorescent dyes provides a distinct emission spectrum for each set of microparticies, allowing the identification of a microparticle set following the pooling of the various sets of microparticies. U.S. Pat. Nos. 6,268,222 and 6,599,331 also are hereby incorporated by reference in their entirety, and in particular for their teachings of various methods of labeling microparticies for multiplex analysis.

5,4.3 Use of Other Methods of Detection

in some embodiments, a separation method may be used to determine feature values for biomarkers in a biomarker profile, such that only a subset of biomarkers within the sample is analyzed. For example, the biomarkers that are analyzed in a sample may be mRNA species from a cellular extract which has been fractionated to obtain on!y the nucleic acid biomarkers within the sample, or the biomarkers may be from a fraction of the total complement of proteins within the sample, which have been fractionated by chromatographic techniques.

Feature values for biomarkers in a biomarker profile can also, for example, be generated by the use of one or more of the following methods described below. For example, methods may inciude nuclear magnetic resonance (NMR) spectroscopy, a mass spectrometry method, such as e!ectrospray ionization mass spectrometry (ESi-MS), ESI-MS/ivIS, ESI-MS/{MS)" (n is an integer greater than zero), matrix-assisted laser desorption ionization time-of-fiight mass spectrometry {MALD I -TOP-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDi-TOF-MS), desorption/ionization on silicon {D!OS}, secondary ion mass spectrometry (SIMS), quadrupole time-of-f!ight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APC!-MS), APCI-MS/MS, APCi-( S)ⁿ, atmospheric pressure photoionization mass spectrometry {APRS-MS}, APPI-MS S, and APPI^«( S)'\ Other mass spectrometry methods may include, inter a!ia, quadrupole, Fourier transform mass spectrometry {FTMS) and ion trap. Other suitable methods ma include chemical extraction partitioning, column chromatography, ion exchange chromatography, hydrophobic (reverse phase) liquid chromatography, isoelectric focusing, one-dimensional polyacrySamide gel electrophoresis (PAGE), two-dimensional poiyacryiamide gel electrophoresis (2D-PAGE) or other chromatography, such as thin-layer, gas or iiquid chromatography, or any combination thereof, in one embodiment, the biological sample may be fractionated prior to application of the separation method.

In one embodiment, iase desorption/ionization time-of-flight mass spectrometry is used to determine feature values in a biomarker profile where th biomarkers ar proteins or protein fragments that have been ionized and vaporized off an immobilizing support by incident Iaser radiation and the feature values are the presence or absence of peaks representing these fragments in th mass spectra profile, A variety of Iaser desorption/ionization techniques are known in the art (see, e.g., Guttrnan et al., 2001 , Anal. Chem. 73:1252-62 and Wei et at, 1999, Nature 399;243-248, each of which is hereby incorporated by reference in its entirety).

Laser desorption/ionization time-of-flight mass spectrometry allows the generation of large amounts of information in a relatively short period of time. A biological sample is applied to one of several varieties of a support that binds all of the biomarkers, or a subset thereof, in the sample. Cell lysafes or samples are directly applied to these surfaces in volumes as small as 0.5 pL, with or without prior purification or fractionation. The !ysates or sample can be concentrated or diluted prior to application onto the support surface. Laser desorption/ionization is then used to generate mass spectra of the sample, or samples, in as little as three hours.

5.4,4 Data Analysis Algorithms

Biomarker expression profiie of T-MSC are factors discriminating between clinical grade

T-MSC and non-clinical grade T-MSC, The identity of these biomarkers and their corresponding features (e.g., expression levels) can be used to develo a decision rule, or plurality of decision rules, that discriminate between clinical grade and non-clinical grade T-MSC. Specific data analysis aigorithms for building a decision rule, or p!uraiity of decision rules, can discriminate between ciinical grade T-MSC and non-clinical grade T-MSC. Once a decision ruie has been built using these exemplary data analysis aigorithms o other techniques known in the art, the decision rule can be used to classify a T-MSC popuiation into one of the two or more phenotypic classes (e.g., a clinical grade or a non-clinical grade T-MSC). This Is accomplished by applying the decision rule to a biomarker profile obtained from the cell culture. Such decision rules, therefore, have enormous value as defining the quality of T-MSC.

In a certain embodiment, provided herein is a method for the evaluation of a biomarker profile from a test cell culture compared to biomarker profiles obtained from a eel! culture in a control population- in some embodiments, each biomarker profile obtained from the control population, as well as the test cell culture, comprises a feature for each of a plurality of different biomarkers. in some embodiments, this comparison is accomplished by (i) developing a decision rule using the biomarker profiles from the control population and (ii) applying the decision rule to the biomarker profile from the test cell culture. As such, the decision rules applied in some embodiments of the present invention are used to determine whether a test cell culture is clinical grade or non-clinical grade, !n certain embodiments, the control population is a c!inical grade T- fVlSC. In other embodiments, the contra! population is BM-MSC.

In some embodiments of the present invention, when the results of the application of a decision rule indicate that the test ceil culture is clinical grade T-MSC, it is used for treatment, !f the results of an application of a decision rule indicate that the test cell culture is non-clinical grade T-MSC, the test cell culture is not used for treatment,

5.5 Modification of T-MSC

Provided herein is a method of modifying mesenchymal stem ceiSs to produce a population of modified MSC that has improved immunosuppressive function. The MSG have the following characteristics; (i) contain >95% of cells expressing group- 1 markers; (ii) contain >80% of cells expressing group 2 markers; (iii) contain <5% of cells expressing group-3 markers; (iv) expresses IL-10 and TGFp; (v) contain <2% of cells expressing !L~8 , IL-12 and TNFa; and (vi) contains <0,001 % of cells co-expressing all group~4 markers, wherein group- 1 markers are CD73, CD90, CD105, CD148, CD166, and CD44, group-2 markers ar CD13, CD29, CD54, CD49E, group-3 markers ar CD45, CD34, CD31 and SSEA4 _: and group-4 markers are OCT4, NANQG, TRA-1 -60 and SSEA4,

Provided herein is a method of increasing immunosuppressive function of T-MSC by increasing the expression of AIF. in an embodiment, the method comprises decreasing the expression of PIF. In an embodiment, the method comprises decreasing the expression of !L6, IL12, TNFa, RAGE and other PIF in T-MSC, In an embodiment, the method comprises increasing the expression of TGFp and IL-10 in T-MSC.

!n certain embodiments, the method comprises genetic and epigenetic modifications of T- MSC that are known in the art. !n certain embodiments, the genetic modification or epigenetic regulation includes, but is not limited to, knockout, small hair pin RNA {"shRNA"), micro RNA miRNA"), non-coding RNA {"ncRNA"), mopho!ino o!igo, decoy RNA, DNA methylation regulation, histone methylation regulation, translation inhibition and/or antibody blocking, in certain embodiments, MSG are modified through transposomss, toll-like receptor itgands, or smali molecules.

in certain embodiments, small molecules are used to target any of the signaling pathway components of SL-6 signaling. In certain embodiments, the target includes, but is not limited to, gp130, STAT3, Cathepsin S, NFkappaB, IRF5. in certain embodiments, IL-12 expression is decreased in T-MSC by activation of the prostaglandin E2 pathway, by increasing intracellular cyclic AMP levels with cAfvlP agonists that include, but are not iimited to, forskoiin, cholera toxin, β1- and f32 adrenoreceptor agonists, by inhibition of the NF-κΒ Rel-8 pathway, by treating T- MSC with apoptotic cells, by treatment with phosphatidylserine, by treatment with butyrate, by treatment with Triptolide or extracts from Tripterygium wiifordii or synthetic forms or Triptolide {i.e., Minnelide).

!n certain embodiments, MSC may be modified to express a certain marker using methods known in the art of recombinant DNA. !n certain embodiments, MSG may be modified by transfection using the nucleotide sequence encoding the marker. The marker can be inserted into an appropriate expression vector, i.e., a vector whic contains the necessary elements for the transcription and translation of the inserted coding sequence. The necessary transcriptional and translational elements can also be present. The regulatory regions and enhancer elements can be of a variety of origins, both natural and synthetic. A variety of host-vector systems may be utilized to express the marker. These include, but are not Iimited to, mammalian ceil systems infected with virus (e.g., vaccinia virus, adenovinis, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA; and stable ceil Sines generated by transformation using a selectable marker. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may b used.

Once a vector encoding the appropriate marker has been synthesized, the MSC is transformed or transfected with the vector of interest.

Standard methods of introducing a nucleic acid sequence of interest into the MSC can be used. Transformation may be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus and transducing a host cell with the virus, and by direct uptake of the polynucleotide. Mammalian transformations {i.e., transfeciions} by direct uptake may be conducted using the calcium phosphate precipitation method of Graham & Van der Eb, 1978, Virol. 52:546, or the various known modifications thereof. Other methods for introducing recombinant polynucleotides into cells, particularly into mammalian ceils, include dextran-mediated transfection, calcium phosphate mediated transfection, po!ybrene mediated transfection, protopiast fusion, electroporation, encapsulation of the poSynucieotide(s} in liposomes, and direct microinjection of the polynucleotides into nuclei, Such methods are well-known to one of skill in the art.

In a preferred embodiment, stable ceil lines containing the constructs of interest are generated for high throughput screening. Such stable eels Sines may be generated by introducing a construct comprising a selectable marker, allowing the eels to grow for 1-2 days in an enriched medium, and then growing the ceils on a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and aiiows ceils to stabiy integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into ceil Sines.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigier, et ai., 1977, Cell 11 :223), hypoxanthine-guanine phosphoribosyStransferase (Szybaiska & Szybalski, 1962, Proc. Natl, Acad. Set. USA 48:2026), and adenine phosphoribosyltransferase (Lowy, et a!., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-celis, respectively. Also, anti-metabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate {Wigler, et aL 1980, Nati. Acad. Sci. USA 77:3567; O'Hare, et a!., 1981 , Proc, Natl, Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenoiic acid (Mulligan & Berg, 1981 , Proc. Nati. Acad. Sci. USA 78:2072); neo, whic confers resistanc to the aminoglycoside G-4 8 (Colberre-Garapin, et al., 1981 , J. ivlol. Biol. 150:1 ); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147) genes.

5,6 Stem Cell Collection Composition

The stem ceil collection composition can comprise any physioiogica!y-accepiafale solution suitable for the collection and/or culture of stem cells, for example, a saline solution (e.g., phosphate-buffered saline, Kreb's solution, modified Kreb's solution, Eagle's solution, 0.9% NaCI. etc.), a culture medium (e.g., D EM, H.DMEM, etc.), and the like.

The stem cell collection composition can comprise one or more components that tend to preserve stem ceils, that is, prevent the stem ceils from dying, or delay the death of the stem ceils, reduce the number of stem ceils in a population of celis that die, or the like, from the time of collection to the time of culturing. Such components can be, e.g., an apoptosis inhibitor (e.g., a caspase inhibitor or JNK inhibitor); a vasodilator (e.g., magnesium sulfate, an antihypertensive drug, atrial natriuretic peptide (ANP). adrenocorticotropin, corticoiropin-reieasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc.); a necrosis inhibitor {e.g., 2-{1 H-lndo!-3- yi)-3-pentylamino-maleimide, pyrrolidine dithiocarbamate, or clonazepam); a TNF-a inhibitor; and/o an oxygen-carrying perf!uorocarbon (e.g., perfluorooctyi bromide, perf!uorodecyl bromide, etc.). The stem ceil collection composition can comprise one or more tissue-degrading enzymes, e.g., a metalloprotease, a serine protease, a neutral protease, an RNase, or a DNase, or the like. Such enzymes inciude, but are not limited to, co!!agenases (e.g., co!iagenase !, if , ill or IV, a collagenase from Clostridium histoiyticum, etc.); dispase, ihermolysin, elastase, trypsin, IIBERASE, hyaiuronidase, and the like.

The stem eel collection composition can comprise a bacteriocidaiSy or bacteriostaticaliy effective amount of an antibiotic, in certain non-limiting embodiments, the antibiotic is a macrolide (e.g., tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime, cefprozii, cefaclor, cefixime or cefadroxi!), a clarithromycin, an erythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g., ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, a streptomycin, etc. In a particular embodiment, the antibiotic is active against Gram(+) and/or Gram(-) bacteria, e.g., Pseudomonas aeruginosa, Staphylococcus aureus, and the like.

The stem cell collection composition can aiso comprise one or more of the following compounds: adenosine (about 1 mM to about 50 mM); D-gSucose (about 20 mM to about 100 mM); magnesium ions (about 1 mivl to about 50 mM); a macromo!ecu!e of mo!ecu!ar weight greater than 20,000 daltons, in one embodiment, present in an amount sufficient to maintain endothelial integrity and celiular viability (e.g., a synthetic o naturally occurring colloid, a polysaccharide such as dextran or a polyethylene glycol present at about 25 g/i to about 100 g i, or about 40 g i to about 60 g/l); an antioxidant (e.g., butyiaied hydroxyanisoie, butyiaied hydroxyioiuene, glutathione, vitamin C or vitamin E present at about 25μΜ to about 100 μΜ}; a reducing agent (e.g. , N-aeetylcysteine present at about 0,1 mM to about 5 mM); an agent that prevents caicium entry into ceils (e.g. , verapamil present at about 2 μΜ to about 25 μΜ); nitroglycerin {e.g., about 0.05g/L to about 0.2g/L); an anticoagulant, in one embodiment, present in an amount sufficient to help prevent dotting of residual blood (e.g., heparin or hirudin present at a concentration of about 1000 units/1 to about 100,000 units/!); or an amiioride containing compound (e.g., amiioride, ethyl isopropyl amiioride, hexamefhyiene amiioride, dimethyl amiioride or isobutyl amiiorid present at about 1.0 μΜ to about 5 μΜ).

5,7 fmmuriomoduiation using T- fSC

Provided herein is the modulation of the activity (e.g. reduced cell proliferation, reduced cell survival, impaired ceil migration to sites of inflammation, reduced ability of the cells to promote or proiong inflammation or enhanced ceil functions that promote the restoration of heaithy tissue or organ homeostasis) of an immune cell, or plurality of immune cells, by contacting the immune cell{s) with a plurality of T-MSC or iT- SC. in one embodiment, the method of modulating an immune response comprises contacting a plurality of immune cells with a plurality of T-!viSC or iT-MSC for a time sufficient for the T-MSC or iT-MSC to detectab!y suppress an immune response, wherein the T-MSC or iT-MSC detectably suppress T celi proliferation in a mixed lymphocyte reaction (MLR) assay.

Since BM-MSC or other adult tissue derived MSG have been used to treat many autoimmune diseases, BM-MSC are also used for tissue repairing by limiting inflammation and secret growth and protective factors, and replacing damaged tissues. As shown later in the examples, T- SC have superior immunosuppressive function to BM-MSC, and thus T-MSC can be used in al! areas and diseases that are currently targeted by BM-MSC.

T-MSC or iPS-MSC used for irnmunornodulation may be derived or obtained from an embryonic stem ceil line or induced piuripotent stem celi line, respectively. T-MSC or iPS-MSC used for irnmunornodulation may also be derived from the same species as the immune cells whose activity is to be modu!ated or from a different species as that of the immune ceils whose activity is to be modulated.

An "immune ceil" in the context of this method means any cell of the immune system, particularly T cells and N (natural killer) celis. Thus, in various embodiments of the method, IT- MSG are contacted with a piuraiity of immune ceiis, wherein the piuraiity of immune cells are, or comprise, a piuraiity of T cells (e.g., a piuraiity of CD3⁺ T ceils, GD4* T celis and/o CDS^* T ceils) and/or natural kilier cells. An "immune response" in the context of the method can be any response by an immune cell to a stimulus normaiiy perceived by an immune ceil, e.g., a response to the presence of an antigen, in various embodiments, an immune response can be the proliferation of T ceils (e.g., CD3' T celis, CD4* T celis and/or CD8⁺ T cells) in response to a foreign antigen, such as an antigen present in a transfusion or graft, or to a self-antigen, as in an autoimmune disease. The immune response can also be a proliferation of T cells contained within a graft. The immune response can also be any activity of a natural kiiler (NK) ceil, the maturation of a dendritic celi, or the like. The immune response can aiso be a local, tissue- or organ-specific, or systemic effect of an activity of one or more classes of immune celis, e.g., the immune response can be graft versus host disease, inflammation, formation of inflammation- related scar tissue, an autoimmune condition (e.g., rheumatoid arthritis, Type I diabetes, lupus erythematosus, etc.), and the like,

"Contacting" in this context encompasses bringing the T-MSC and immune cells together in a single container (e.g., culture dish, flask, via!, etc.) or in vivo, for example, the same individual {e.g., mammai, for example, human). In a preferred embodiment, the contacting is for a time sufficient, and with a sufficient number of T-MSC and immune cells, that a change in an immune function of the immune ceiis is detectable. More preferably, in various embodiments, the contacting is sufficient to suppress immune function (e.g., T cell proliferation in response to an antigen) by at ieas! 50%, 60%, 70%, 80%, 90% or 95%, compared to the immune function in the absence of the T-MSC. Such suppression in an in vivo context can be determined in an in vitro assay; that is, the degree of suppression in the in vitro assay can he extrapolated, for a particular number of T-MSC and a number of immune cells in a recipient individual, to a degree of suppression in the individual.

The invention in certain embodiments provides methods of using T- SC to modulate an immune response, or the activity of a plurality of one or more types of immune cells, in vitro, Contacting the T-MSC and plurality of immune cells can comprise combining the T-MSC and immune cells in the same physical space such that at least a portion of the plurality of T-MSC interacts with at least a portion of the plurality of immune cells; maintaining the T-MSC and immune ceils in separate physical spaces with common medium; or can comprise contacting medium from one or a culture of T-MSC or immune cells with the other type of cell (for example, obtaining culture medium from a culture of T-MSC and resuspending isolated immune cells in the medium). In a specific example, the contacting is a Mixed Lymphocyte Reaction {MLR},

Such contacting can, for example, take place in an experimental setting designed to determine the extent to which a particular plurality of T-MSC is immunomodulatory, e.g., immunosuppressive. Such an experimental setting can be, for example, a mixed lymphocyte reaction (MLR) or regression assay. Procedures fo performing the MLR and regression assays are well-known in the art. See, e.g. , Schwarz, "The Mixed Lymphocyte Reaction^': An In Vitro Test fo Tolerance," J. Exp. Med. 127(5):879~890 (1968); Lacerda et ai., "Human Epstein-Barr Virus (EBV Specific Cytotoxic T Lymphocytes Home Preferentially to and Induce Selective Regressions of Autologous EBV-induced B Lymphoproliferations in Xenografted C.B-17 Scid/Scid Mice," J. Exp, Med. 183:1215-1228 (1996), In a preferred embodiment, an MLR is performed in which a plurality of T-MSC ar contacted with a plurality of immune cells (e.g., lymphocytes, for example, CDS^1' CD4* and/or CDS* T lymphocytes).

The MLR can be used to determine the immunosuppressive capacity of a plurality of T- MSC. For example, a plurality of T-MSC can be tested in an MLR comprising combining CD4~ or CD8^* T celis, dendritic celis (DC) and T-MSC in a ratio of about 10:1 ;2. wherein the T celis are stained with a dye such as, e.g., CFSE that partitions into daughter cells, and wherein the T ceils are allowed to pro!iferate for about 6 days. The plurality of T-MSC is immunosuppressiv if the T cell proliferation at 6 days in the presence of T-MSC is detectably reduced compared to T ceil proliferation in the presence of DC and absence of T-MSC, in such an MLR, T-MSC are either thawed or harvested from culture. About 10,000 T-MSC are resuspended in 100 μί of medium (RPMI 1640, 1 mM HEPES buffer, antibiotics, and 5% pooled human serum), and allowed to attac to the bottom of a we!! for 2 hours. CD4^* and/or CD8⁺ T cells are isolated from whole peripheral blood mononuclear celis with Mi!tenyi magnetic beads. The cells are CFSE stained, and a total of 100,000 T ceils (CD4^* T ceils alone, CDS* T celis alone, or equal amounts of CD4* and CD8* T celis) are added per weli. The volume in the we!! is brought to 200 μΙ, and the MLR is ai!owed to proceed. in one embodiment, therefore, the invention provides a method of suppressing an immune response comprising contacting a plurality of immune ceils with a plurality of T-MSC for a time sufficient for the T-MSC to deteetabiy suppress T ce!i proliferation in a mixed lymphocyte reaction (MLR) assay.

Populations of T-MSC obtained from different embryonic stem cell lines, can differ in their ability to modulate an activity of an immune cell, e.g., can differ in their abiiity to suppress T cell activity or proliferation or NK cell activity. It is thus desirable to determine, prior to use, the capacity of a particular population of T-MSC for immunosuppression. Such a capacity can be determined, for example, by testing a sample of the stem ceil population in an MLR or regression assay, !n one embodiment, an MLR is performed with the sample, and a degree of immunosuppression in the assay attributable to the T-MSC is determined. This degre of immunosuppression can then be attributed to the stem cell population that was sampled. Thus, the MLR can be used as a method of detesmining the absolute and relative ability of a particular population of T-MSC to suppress immune function. The parameters of the MLR can be varied to provide more data or to best determine the capacity of a sample of T-MSC to immunosuppress. For exampie, because immunosuppression by T-MSC appears to increase roughly in proportion to the number of T-MSC present in the assay, the MLR can be performed with, in one embodiment, two or more numbers of stem celis, e.g., 1 x 1Q³, 3 x 10³, 1 x 10⁴ and/or 3 x 10⁴ T- MSC per reaction. The number of T-MSC relative to the number of T cells in the assay can also be varied. For example, T-MSC and T ceils in the assay can be present in any ratio of, e.g., about 10:1 to about 1 :10, preferably about 1 :5, though a relatively greater number of T-MSC or T cells can be used.

The invention also provides methods of using T-MSC to modulate an immune response, or the activity of a plurality of one or more types of immune cells, in vivo. T-MSC and immune celis can be contacted, e.g., in an individual that is a recipient of a plurality of T-MSC, Where the contacting is performed in an individual, in one embodiment, the contacting is between exogenous T-MSC (that is, T-MSC not derived from the individual) and a plurality of immune cells endogenous to the individual. In specific embodiments, the immune cells within the individual are CDS ^' T cells, CD * T cells, CDS' T cells, and/or NK cells.

Such immunosuppression using T-MSC would be advantageous for any condition caused or worsened by, o related to, an inappropriate or undesirabie immune response. T-MSC- mediafed immunomodulation, e.g., immunosuppression, wouid, for exampie, be useful in the suppression of an inappropriate immune response raised by the individual's immune system against one or more of its own tissues. In various embodiments, therefore, the invention provides a method of suppressing an immune response, wherein the immune response is an autoimmune disease, e.g., lupus erythematosus, diabetes, rheumatoid arthritis, or multiple sclerosis. The contacting of the plurality of T-MSC with the plurality of one or more types of immune ceils can occur in viva in the context of, or as an adjunct to, for example, grafting or transplanting of one or more types of tissues to a recipient individual. Such tissues may be, for example, bone marrow or blood; an organ; a specific tissue (e.g., skin graft); composite tissue allograft {i.e., a graft comprising two or more different types of tissues); etc. in this regard, the T-MSC can be used to suppress one or more immune responses of one or more immune ceils contained within the recipient individual, within the transplanted tissue or graft, or both. The contacting can occur before, during and/or after the grafting or transplanting. For example, T-MSC can be administered at the time of the transplant or graft. The T-MSC can aiso, o alternatively, be administered prior to the transplanting or grafting, e.g., about 1 , 2, 3, 4, 5, 6 or 7 days prior to the transplanting or grafting, T-MSC can also, or alternatively, be administered to a transplant or graft recipient after the transplantation or grafting, for example, about 1 , 2, 3, 4, 5, 8 or 7 days after the transplanting or grafting. Preferably, the plurality of T ceiis are contacted with the plurality of T-MSC before any detectable sign or symptom of an immune response, eithe by the recipient individual or the transplanted tissue or graft, e.g. , a detectable sign or symptom of graft- versus-host disease o detectable inflammation, is detectable.

In another embodiment, the contacting within an individual is primarily between exogenous T-MSC and exogenous progenitor cells or stem cells, e.g., exogenous progenitor cells o stem cells that differentiate into immune cells. For example, individuals undergoing partial or full immunoabiation or mye!oabiation as an adjunct to cancer therapy can receive T-MSC in combination with one or more other types of stem or progenitor cells. For example, the T-MSC can be combined with a plurality of CD34^! ceiis, e.g., CD34^! hematopoietic stem cells. Such CD34⁺ cells can be, e.g., CD34^* cells from a tissue source such as peripheral blood, umbilical cord b!ood, placental blood, or bone marrow. The CD34⁺ cells can be isolated from such tissue sources, or the whole tissue source (e.g., units of umbilical cord blood or bone marrow) or a partially purified preparation from the tissue source {e.g., white blood cells from cord blood) can be combined with the T-MSC.

The T-MSC are administered to the individual preferably in a ratio, with respect to the known or expected number of immune ceils, e.g. , T cells, in the individual, of from about 10:1 to about 1 :10, preferably about 1 :5, However, a plurality of T-MSC can be administered to an individual in a ratio of in non-limiting examples, about 10,000:1 , about 1 ,000:1 , about 100:1 , about 10:1 , about 1 ;1 , about 1 :10, about 1 :100, about 1 :1 ,000 o about 1 :10,000, Generally, about 1 x 10⁵ to about 1 x 1Q⁸ T-MSC per recipient kilogram, preferably about 1 x 10⁶ to about 1 x 10⁷ T-MSC recipient kilogram can be administered to effect immunosuppression. In various embodiments, a plurality of T-MSC administered to an individual or subject comprises at least, about, or no more than, 1 x 10^s, 3 x 10^s, 1 x 10^s, 3 x 10^δ, 1 x 10⁷, 3 x 10^?, 1 x 10⁸, 3 x 10^s, 1 x 10^s, 3 x10^s T-MSC, or more. The T-MSC can aiso be administered with one or more second types of stem ceils, e.g., mesenchymal stem ceils from bone marrow. Such second stem cells can be administered to an individual with T-MSC in a ratio of, e.g., about 1 ; 10 to about 10.1 ,

To facilitate contacting the T-MSC and immune cells in two, the T-MSC can be administered to the individual by any route sufficient to bring the T-MSC and immune cells into contact with each other. For example, the T-MSC can be administered to the individual, e.g., intravenously, intramuscularly, infraperitoneally, or directly into an organ, e.g. , pancreas. For in vivo administration, the T-MSC can be formulated as a pharmaceutical composition.

The method of immunosuppression can additionally comprise the addition of one or more immunosuppressive agents, particularly in the in vivo context. In one embodiment, the plurality of T-MSC are contacted with the plurality of immune cells in vivo in an individual, and a composition comprising an immunosuppressive agent is administered to the individual. Immunosuppressive agents are well known in the art and include, e.g., anti-T ceii receptor antibodies (monoclonal or polyclonal, or antibody fragments or derivatives thereof), anti-IL-2 receptor antibodies (e.g., Sasiiiximafo (SiMULECT^®) or dac!izumab (ZENAPAX^®), anti T celi receptor antibodies (e.g. , Muromonafo-CD3}, azathioprine, corticosteroids, cyciosporine, tacrolimus, mycophenoiate mofetil, siroiimus, caScineurin inhibitors, and the like. In a specific embodiment, the immunosuppressive agent is a neutralizing antibody to macrophage inflammatory protein (MIP)- 1a or !Ρ-1 β.

5,8 Preservation of T-MSC and/or T 8SC-DL

T-MSC and/or T-MSC-DL can be preserved, that is, placed under conditions that allow for long-term storage, or conditions that inhibit celi death by, e.g., apoptosis or necrosis. T-MSC and/or T-MSC-DL can be preserved using, e.g., a composition comprising an apoptosis inhibitor, necrosis inhibitor, in one embodiment, the invention provides a method of preserving a population of stem ceils comprising contacting a population of stem cells with a stem ceil collection composition comprising an inhibitor of apoptosis, wherein the inhibitor of apoptosis is present in an amount and for a time sufficient to reduce or prevent apoptosis in the population of stem ceISs, as compared to a population of stem cells not contacted with the inhibitor of apoptosis. in a specific embodiment, the inhibitor of apoptosis is a caspase inhibitor, in another specific embodiment, the inhibitor of apoptosis is a JNK inhibitor, in a more specific embodiment, the JNK inhibitor does not modulaie differentiation or proliferation of fhe stem cells, in another embodiment, the stem ceil collection composition comprises an inhibitor of apoptosis and an oxygen-carrying perfluorocarbon in separate phases. In another embodiment, the stem celi collection composition comprises an inhibitor of apoptosis and an oxygen-carrying perfluorocarbon in an emulsion, in another embodiment, the stem celi collection composition additionally comprises an emuisifier, e.g., lecithin. In another embodiment, the apoptosis inhibito and the perfiuorocarbon are between about 0°C and about 25 °C at the time of contacting the stem cells, in another more specific embodiment, the apoptosis inhibitor and the perfiuorocarbon are between about 2°C and 10°C, o between about 2°C and about 5 C, at the time of contacting the stem ceils. In another more specific embodiment, the contacting is performed during transport of the population of stem cells. In another more specific embodiment, the contacting is performed during freezing and thawing of the population of stem cells.

in another embodiment, the invention provides a method of preserving a population of T- IvISC and/or T-MSC-DL comprising contacting the population of stem cells with an inhibitor of apoptosis and an organ-preserving compound, wherein the inhibitor of apoptosis is present in an amount and for a time sufficient to reduce or prevent apoptosis in the population of stem cells, as compared to a population of stem cells not contacted with the inhibitor of apoptosis.

Typically, during T-MSC and/or T-MSC-DL collection, enrichment and isolation, it is preferable to minimize or eliminate cell stress due to hypoxia and mechanical stress. In another embodiment of the method, therefore, a stem ceil, or population of stem cells, is exposed to a hypoxic condition during collection, enrichment o isolation for less than six hours during the preservation, wherein a hypoxic condition is a concentration of oxygen that is less than norma! biood oxygen concentration, in a more specific embodiment, the population of stem ceils is exposed to the hypoxic condition for less than two hours during the preservation, in another more specific embodiment, the population of stem cells is exposed to the hypoxic condition for less than one hour, or less than thirty minutes, or is not exposed to a hypoxic condition, during collection, enrichment or isolation. In another specific embodiment, the population of stem cells is not exposed to shear stress during collection, enrichment o isolation.

The T-MSC and/or T-MSC-DL can be cryopreserved, e.g. , in cryopreservation medium in smali containers, e.g., ampoules. Suitable cryopreservation medium includes, but is not limited to, culture medium including, e.g., growth medium, or cell freezing medium, for example commerciafiy available ce!f freezing medium, e.g., C2695, C2639 or C6039 (Sigma), Cryopreservation medium preferably comprises DMSO {dimethyisuifoxide}, at a concentration of, e.g. , about 10% (v/v). Cryopreservation medium may comprise additional agents, for example, methylcellulose and/or glycerol. T-MSC and/or T-MSC-DL are preferably cooled at about 1°C/min during cryopreservation. A preferred cryopreservation temperature is about -80°C to about - 180°C, preferably about -125°C to about -140°C Cryopreserved ceils can be transferred to liquid nitrogen prior to thawing for use, in some embodiments, for example, once the ampoules have reached about -90°C, they are transferred to a liquid nitrogen storage area. Cryopreserved cells preferably are thawed at a temperature of about 25°C to about 40°C, preferably to a temperature of about 37°C, 5.9 Cryopreserved T-MSC and/or T-MSC-DL

The T-MSC and/or T-MSC-DL disclosed herein can be preserved, for example, cryopreserved for later use. Methods for cryopreservation of cells, such as stem ceils, are well known in the art, T-MSC and/or T-MSC-DL can be prepared in a form that is easily administrable to an individual. For example, provided herein are T-MSC and/or T-MSC-DL that are contained within a container that is suitable for medical use. Such a container can be, for example, a sterile plastic bag, flask, jar, or other container from which the T-MSC and/or T-MSC-DL can be easily dispensed. For example, the container can be a biood bag or other plastic, medicaly-acceptable bag suitable for the intravenous administration of a liquid to a recipient. The container is preferably one that allows for cryopreservation of the combined stem cell population. Cryopreserved T-MSC and/or T-MSC-DL can comprise T-MSC and/or T-MSC-DL derived from a single donor, or from multiple donors. The T-MSC and/or T-MSC-DL can be completely HLA- matc ed to an intended recipient, or partially or completely HLA-mismatched.

In another specific embodiment, the container is a bag, flask, or jar. !n a more specific embodiment, the bag is a sterile plastic bag. In a more specific embodiment, the bag is suitable for, allows or facilitates intravenous administration of the T-MSC and/or T-MSC-DL. The bag can comprise multiple lumens o compartments that are interconnected to aliow mixing of the T-MSC and/or T-MSC-DL and one or more other solutions, e.g.. a drug, prio to, or during, administration, in another specific embodiment, the composition comprises one or more compounds that facilitate cryopreservation of the combined stem ceil population, in another specific embodiment, the T-MSC and/or T-MSC-DL is contained within a physiologically- acceptable aqueous solution. In a more specific embodiment, the physiologicaiiy-acceptahie aqueous solution is a 0.9% NaCI solution, in another specific embodiment, the hES-MSC are HLA-matched to a recipient of the stem cell population. In another specific embodiment, the combined stem cell popuSation comprises hES-MSC that are at least partially HLA-mismatched to a recipient of the stem ceil population.

5,10 Differentiation of T-MSC into MuJtipSe Lineages

T-MSC may be differentiated into various cell lineages including neuronal lineage cells o neurons, or adipocytes, or myoblasts, or fibroblasts, or osteoblasts or chrondrocytes. Unless specifically indicated, T-MSC may be plated onto ceil culture plates coated with gelatin, collagen, fibronectin, Matrigel, iaminin, vitronectin, or poly(lysine). T-MSC may be plated at a concentration of 1 10³ ceils/cm² to 1 * 10⁴ ceils/on² in serum free medium or serum-containing medium with bovine serum FBS or ABHS, T-MSCs plated according to the above mentioned conditions may be differentiated by one of the following methods.

In one embodiment, T-MSC may be differentiated in medium containing 1-50 ng/mL Fibroblast Growth Factor {FGF}-2 {optimally 10 ng/ml) plus 1-50 ng/mi Epidermal Growth Factor (EGF) {optimally 10 ng/ml) plus 0.5-5 ng/ml Platelet-Derived Growth Factor (PDGF) (optimally 1 ng/ml). The medium is changed every 2 io 3 days and the celis are harvested after 2 -4 weeks with an expected yieid of 0.5 x 10^e ~ 2 x 10^e neuronal lineage cells per 1 x 10^s T-MSC,

in another embodiment, T-MSC may be differentiated into neuronal lineage cells by plating on Poiy-l-omithine and laminin coated plates, T-MSCs will be differentiated in three stages. Stage 1: 1-50 ng/mi FGF-2 (optimally 10 ng/rrs!) and 1-50 ng/mi EGF (optimally 10 ng/rrs!), to prime hMSCs towards a neural fate. Stage 2: 10-200 ng/ml Sonic Hedgehog (SHH) (optimali 100 ng/mi), 1-50 ng/mi FGF-S (human) (optimally 10 ng/ml) and 50-500 Μ AAP (optimally 200 μΜ), for initiating midbrain specification. Stage 3; 5-500 ng/ml Giia!-Derived Neurotrophic Factor (GDNF) {optimally 50 ng/ml) and 50-500 μ AAP (optimaliy 200 μΜ), for inducing differentiation and maturation towards a dopaminergic neuronal phenotype. Each stage is applied for 1 week and the adherent cells are passaged by disassociation with Trypsin or TrypLE/dispase between eac stage. Growth factors are replenished every day and the medium is changed every 2 days. Expected yield is 0.5 x 10⁶~ 4 x 10⁶ neuronal lineage cells per 1 x 10⁶ T-MSC.

In another embodiment, T-MSC may be differentiated into neuronal iineage ceils in

Neurobasa! medium (Gi co) containing 0.25 x B-27 supplement plus 10-200 ng/ml Sonic Hedgehog (SHH) (optimally 100 ng/mi), plus 1-50 ng/mi FGF-8 (mouse) (optimaliy 0 ng/ml) plus 1-200 ng/mi FGF-2 {opiimaily 50 ng/ml). Cells are harvested after 6~ and 12-days. Media is not replaced during this period. Expected yield is 0.5 x 10⁶~ 4 x 10^s neuronal Iineage celis per 1 x 10⁸ T-MSC.

In another embodiment, T-MSC may be differentiated into neuronal iineage eels in two stages. Stage 1 ; T-MSC are cultured in serum-free medium (DMEM) supplemented with 2 mM giutarnine, 1-20 U/ml (optimally 12.5 U/mi) nystatin, N2 supplement, and 2-50 ng/mi {optimally 20 ng/mi) fibroblast growth facior-2 (FGF-2) and 1 -50 ng/mL EGF (optimally 10 ng/mi) for 48-72 hours. Stage 2: cells are cultured in Neurobasa! medium plus B27 supplement plus 0,1-10 mM (optimaliy 1 mM) dibutyryl cyclic AMP (dbcAMP), 3-isobutyi-1-methylxanthine (IBMX), and 10- 500 uM (opiimaily 200 μΜ) ascorbic acid plus 1-100 ng/mi BDNF (optimally 50 ng/ml), 1-50 ng/ml gliaS-derived neurotrophic factor (GDNF; optimally lOng/m!), 0.2-10 ng/mi transforming growth factor-j&S (TGF- S3, optimally 2 ng/mi), and 0.05-5 μΜ all-fransretinoie acid (RA, optimaliy 0.1 μΜ). Each stage is applied for 1 week and the adherent celis are passaged by disassociation with Trypsin or TrypLE/dispase between each stage. The medium is changed every 2 days and the expected yield is 0.5 x 10⁶ - 4 x 10⁶ neuronal Iineage cells per 1 x 10° T-MSC.

In another embodiment, T-MSC may be cultured to induce osteogenic differentiation. T- MSCs will be cultured in low giucose DMEM plus 10% FCS, 1 -150uM (optimally 80 μΜ) ascorbic acid 2- phosphate, 0.5-5 μΜ (optimaliy 1 μΜ) dexamethasone, and 1-100 mM (optimally 20 mM) beta-g!ycerophosphate. The medium is changed every 2 to 3 days and the expected yield is 0.5 x 10^e-4 x 10° neuronal lineage ceils per 1 x 10^s T-MSC after 2 weeks. in another embodiment, T-MSC may be cu!tured to induce adipogenic differentiation. T- MSCs wiii be grown in low glucose D E plus 20% FCS, 1 -10 g/rnl (optimaiiy 5 g mi) insulin, 0,5-10 μΜ (optimaiiy 2 μΜ) dexamethasone, 0.1-1 mM (optimally 0.5 mM)

isobutySmethylxanthine, and 1 -100 μΜ (optimaiiy 60 μ ) indomethacin. The medium is changed every 2 to 3 days and the expected yield is 0.5 x 10⁶- 4 x 10⁶ neuronal lineage ceiis per 1 x 10⁶ T-MSC after 4 weeks.

in another embodiment, T-MSC may be cultured to induce chondrogenic differentiation. T- SC wiii be grown in a pellet in high giucose DMEM supplemented with 0.5-10 mM (optimally 1 mM) Sodium Pyruvate, 0.05-1 mM (optimaliy 0.1 mM) ascorbic acid 2 -phosphate, 0.05-1 μΜ (optimaiiy 0,1 μΜ) dexamethasone, 0,2-2% (optimaliy 1 %) ITS, and 1-50 ng/mi (optimaiiy 10 ng/mL) TGF~p3, The medium is changed every 2 to 3 days and the expected yield is 0.5 x 10^e-

4 x 10* neuronal lineage cells per 1 x 10⁶ T-MSC after 20 days.

in another embodiment, T-MSC may be cultured to induce myogenic differentiation. T- MSC will be grown in Sow-glucose DMEM supplemented with 10% FBS, 1-20 μΜ (optimaliy 10 μΜ) 5-azacytidine, and 1-50 ng/mi (optimaiiy 10 ng/ml) basic FGF. After 24 hours, the myogenic induction medium wiii be replaced with DMEM supplemented with 10% FBS plus 1-50 ng/mi (optimally 10 ng/mi) basic FGF. The medium is changed every 2 to 3 days and the expected yield is 0,5 x 10^e - 4 x 10^s neuronal iineage cells per 1 x 10⁶ T-MSC after 2 weeks.

In another embodiment, T-MSC may be cultured to induce fibroblast differentiation. T- MSG wiii be grown in hMSCs that were treated with DMEM plus 10% FBS supplemented 50-200 ng/mi (optimaiiy 100 ng/mi) of recombinant human Connective Tissue Growth Factor (CTGF) and 1-100 μρ,/mi (optimaiiy 50 pg/m!) ascorbic acid. The medium is changed every 3 to 4 days and the expected yield is 0.5 x 0^s - 4 x 10^s neuronal Iineage cells per 1 x 10^δ T-MSC after 4 weeks.

All the ceil lineages and cell types derived from T-MSC using any differentiation methods including, but not limited to, the methods above are called T-MSC-DL throughout.

5,11 Pharmaceuticai Preparations

In one embodiment, provided herein is a pharmaceutica! composition comprising a therapeutically effective amount of a T-MSC and a pharmaceutically acceptable carrier.

The pharmaceutical compositions can comprise any number of T-MSC and/or T-MSC-

DL For example, a single unit dose of T-MSC can comprise, in various embodiments, about, at least, or no more than 1 x 10^s, 5 x 10^s, 1 x 10^δ ₍ 5 x 10⁶, 1 x TO⁷, 5 x 10⁷, 1 x 10^s, 5 x 10^s, 1 x 10^s,

5 x 10³, 1 x 10^1Q, 5 x 10""^', 1 x 10" or more T-MSC and/or T-MSC-DL.

The pharmaceutica! compositions disclosed herein comprise popuiations of cells that comprise 50% viable celis or more (that is, at least 50% of the ceiis in the population are functional or Ssving). Preferably, at ieast 60% of the ceiis in the population are viable. More preferably, at least 70%, 80%, 90%, 95%, or 99% of ihe cells in the population in the pharmaceutical composition are viable.

The pharmaceuticai compositions disclosed herein can comprise one or more compounds that, e.g., facilitate engraftment (e.g., anti-T-cel! receptor antibodies, an immunosuppressant, or the like); stabilizers such as albumin, dextran 40, gelatin, hydroxyethyS starch, and the like.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions thai are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human, and approved by a regulatory agency of a Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans, "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceuticai carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanoS, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, cachets, troches, lozenges, dispersions, suppositories, ointments, cataplasms (poultices), pastes, powders, dressings, creams, plasters, patches, aerosols, gels, liquid dosage forms suitable for parenteral administration to a patient, and steriie solids (e.g. , crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable form of carrier so as to provide the form for proper administration to the patient. The formulation should suit th mode of administration.

Pharmaceutical compositions adapted for oral administration may be capsules, tablets, powders, granules, solutions, syrups, suspensions (in non-aqueous or aqueous liquids), or emulsions. Tablets or hard gelatin capsules may comprise lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatin capsules may compris vegetable oils, waxes, fats, semi-solid, o liquid poiyois. Solutions and syrups may comprise water, po!yo!s, and sugars. An active agent intended for oral administration may be coated with or admixed with a material that delays disintegration and/or absorption of the active agent in the gastrointestinal tract. Thus, the sustained release may be achieved over many hours and if necessary, the active agent can be protected from degradation within the stomach. Pharmaceutical compositions for oral administration may be formuiated to facilitate release of an active agent at a particular gastrointestinal location due to specific pH or enzymatic conditions.

Pharmaceutical compositions adapted for transdermal administration may be provided as discrete patches intended to remain in intimate contact with the epidermis of the recipient over a prolonged period of time.

Pharmaceutical compositions adapted for nasal and puimonary administration may comprise solid carriers such as powders which can be administered by rapid inhalation through the nose. Compositions for nasal administration may comprise liquid carriers, such as sprays or drops. Alternatively, inhalation directly through into the iungs may be accomplished by inhalation deeply or installation through a mouthpiece. These compositions may comprise aqueous or oii solutions of the active ingredient. Compositions for inhalation may be supplied in specially adapted devices including, but not limited to, pressurized aerosols, nebulizers or insufflators, which can be constructed so as to provide predetermined dosages of the active ingredient.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injectable solutions or suspensions, which may contain anfi-oxidants, buffers, faacteriostats, and solutes that render the compositions substantially isotonic with the blood of the subject. Other components which may be present in such compositions inciude water, alcohols, poiyols, glycerine, and vegetable oils. Compositions adapted for parental administration may be presented in unit-dose or muiti-dose containers, such as sealed ampules and vials, and may b stored in a freeze-dried {lyophized) condition requiring only the addition of a sterile carrier, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Suitable vehicles that can be used to provide parenteral dosage forms of the invention are weli known to those skilied in the art. Examples inciude: Water for Injection USP; aqueous vehicles such as Sodium Chloride Injection, Ringer's injection, Dextrose injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscibSe vehicles such as ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Selection of a therapeutically effective dose will be determined by the skilled artisan considering several factors which wiii be known to one of ordinary skill in the art. Such factors inciude the particular form of the inhibitor, and its pharmacokinetic parameters such as bioavailability, metabolism, and haif-iife, which will have been established during the usual development procedures typicai!y employed in obtaining regulatory approval for a pharmaceuiicai compound. Further factors in considering the dose include the condition or disease to b treated or the benefit to be achieved in a normal individual the body mass of the patient, the route of administration, whether the administration is acute or chronic, concomitant medications, and other factors well known to affect the efficacy of administered pharmaceutical agents. Thus, the precise dose should be decided according to the judgment of the person of skill in the art, and each patient's circumstances, and according to standard clinical techniques. in certain embodiments, patients are treated with antipyretic and/or antihistamine (acetaminophen and diphenhydramine hydrochloride) to minimize any possible DMSO infusion toxicity related to the cryopreserve component in the hES-MSC treatment.

5.12 T-MSC Conditioned Media and Derivatives

The T-MSC disclosed herein can be used to produce conditioned medium that is immunosuppressive, that is, medium comprising one or more biomolecules secreted or excreted by the stem cells that have a detectable immunosuppressive effect on a plurality of one or more types of immune cells, !n various embodiments, the conditioned medium comprises medium in which T-MSC have grown for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or more days, in other emboditnenis, the conditioned medium comprises medium in which T-MSC have grown to at least 30%, 40%, 50%, 60%, 70%, 80%, 90% confluence, or up to 100% confluence. Such conditioned medium can be used to support the culture of a separate population of T-MSC, or stem ceils of another kind. In another embodiment, the conditioned medium comprises medium in which T-MSC have been differentiated into an adult cell type, in another embodiment, the conditioned medium of the invention comprises medium in which T-MSC and non-T-MSC have been cultured.

Thus, in one embodiment, the invention provides a composition comprising culture medium, ceil lysate and/or other derivatives from a culture of T-MSC, wherein the T-MSC (a) adhere to a substrate; (b) express CD73, CD105, CD90, CD29, CD44, CD146, 11-10, TGFb2, HGF, but do not express IL-6, T Fa, !L-12 and/or RAGE. In another specific embodiment, the composition comprises an anti-proliferative agent, e.g., an anti-MiP-1a or anti-MSP-Ι β antibody.

Provided herein is a method of using T-MSC as described herein as feeder ceils for bone marrow hematopoietic stem cell, peripheral blood hematopoietic stem cell and umbilical-cord hematopoietic stem cell expansion. In certain embodiments, the T-MSC suitable for the disclosed method express Stro-3, Stro-1 , DL1 , and/or Nestin. The T-MSC can also b modified or engineered to express high level of Stro-3, Stro-1 , DL1 , Nestin or Frizzle using the method disclosed herein in Section 5.5. In certain embodiments, T-MSC is co-cultured with bone marrow hematopoietic stem cells, peripheral biood hematopoietic stem ceils and/or umbilical -cord hematopoietic stem cells, in certain embodiments, the T-MSC are mesenchymal stromal cells. Provided herein is a co-culture of T-MSC as described herein and bone marrow hematopoietic stem ce!is. Provided herein is a co-culture of T-MSC as described herein and umbiiicai-cord hematopoietic stem ceils,

5J3 Matrices Comprising T-MSC and/or T-MSC Derived Lineages The invention further comprises matrices, hydrogeis, scaffolds, and the like that comprise

T-MSC and/or T-WSC-DL T-MSC and/or T- SC-DL can be seeded onto a natural matrix, e.g., a biomateriaS. in certain embodiments, the scaffold is obtained by 3D printing. The T-MSC and/or T-MSC-DL can be suspended in a hydrogei solution suitable for, e.g., injection. Suitable hydrogeis for such compositions include seSf-assembling peptides, such as RAD16. In one embodiment, a hydrogei solution comprising the ceils can be allowed to harden, for instance in a moid, to form a matrix having cells dispersed therein for implantation. T-MSC and/or T-MSC-DL in such a matrix can aiso be cultured so that the celis are mitoticaliy expanded prior to implantation. The hydrogei is, e.g., an organic poiyrner (natural or synthetic) that is cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure that entraps water molecules to form a gel. Hydrogel-forming materials include polysaccharides such as alginate and salts thereof, peptides, polyphosphazines, and polyacryiates, which are cross- linked ionicaily, or block polymers such as polyethylene oxide-polypropylene glycol block copolymers which are cross-linked by temperature or pH. respectively, in some embodiments, the hydrogei or matrix of the invention is biodegradable. In some embodiments of the invention, the formulation comprises an in situ polymerizabie gel {see, e.g., U.S. Patent Application Publication 2002/0022676; Anseth et aS., J, Control Release, 78(1 -3};199-209 (2002); Wang et ai„ Biomateriais, 24(22):3969~8Q (2003).

in some embodiments, the polymers are at least partially soiuble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, that have charged side groups, or a monovalent ionic salt thereof. Examples of polymers having acidic side groups that can be reacted with cations are poly(phosphazenes), pofy(acrylic acids), pofy(methacryiic acids), copolymers of acrylic acid and methacryiic acid, poly(vinyi acetate), and sulfonated polymers, such as sulfonated polystyrene. Copolymers having acidic side groups formed by reaction of acrylic or methacryiic acid and viny! ether monomers or polymers can aiso be used. Examples of acidic groups are carboxylic acid groups, sulfonic acid groups, haiogenated (preferably fiuorinated) alcohol groups, phenolic OH groups, and acidic OH groups.

The T-MSC, T-MSC-DL and/or co-cultures thereof can be seeded onto a three- dimensional framework or scaffold and implanted in vivo. Such a framework can be implanted in combination with any one or more growth factors, celis, drugs or other components thai stimulate tissue formation or otherwise enhance or improve the practice of the invention.

Examples of scaffolds that can be used in the present invention include nonwoven mats, porous foams, or self-assembSing peptides, Nonwoven mats can be formed using fibers comprised of a synthetic absorbable copolymer of g!ycolic and lactic acids {e.g... PGA/PLA) (VICRYL, Eihicon, Inc., Somerv!e, N.J.). Foams, composed of, e.g., poly{s- caproiactone)/poly(glyco!ic acid) (PGL/PGA) copolymer, formed by processes such as freeze- dryi g, or lyophilizafion {see, e.g., U.S. Pat, No, 6,355,699), can also be used as scaffolds,

The T-MSC and/or T-!VSSC-DL can also be seeded onto, or contacted with, a physio!ogicai!y-acceptab!e ceramic materia! including, but not limited to, mono-, di~, tri-, aipha-tri-, beta-tri-_; and tetra-ca!cium phosphate, hydroxyapatite, fluoroapatites, calcium sulfates, calcium fluorides, calcium oxides, calcium carbonates, magnesium calcium phosphates, biologically active glasses such as BIOGLASS^8,1, and mixtures thereof. Porous biocompatible ceramic materials currently commercially available include SURGSBONE^® (Can edica Corp., Canada), END080N^¾> {Merck Biomateriai France, France), CEROS^® (Mathys, AG, Beft!ach, Switzerland), and mineralized collagen bone grafting products such as HEALOS™ (DePuy, inc., Raynham, Mass.) and ViTOSS^®, RHAKOSS™, and CORTOSS^® (Orthovita, Malvern, Pa.). The framework can be a mixture, biend or composite of natural and/or synthetic materials.

In anothe embodiment, T-MSG and/or T-MSC-DL can be seeded onto, or contacted with, a feit, which can be, e.g., composed of a multifilament yarn made from a bioabsorbab!e material such as PGA, PLA, PCL copolymers or blends, or hyaluronic acid.

The T-MSC and/or T-MSC-DL can, in another embodiment, be seeded onto foam scaffolds that may be composite structures. Such foam scaffolds can be molded into a useful shape, such as that of a portion of a specific structure in the body to be repaired, replaced or augmented, in some embodiments, the framework is treated, e.g., with 0.1 acetic acid followed by incubation in poiyiysine, PBS, and/or collagen, prior to inoculation of the cells of the invention in order to enhance cell attachment. External surfaces of a matrix may be modified to improve the attachment or growth of cells and differentiation of tissue, such as by plasma-coating the matrix, or addition of one or more proteins (e.g., collageos, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin- -sulfate, chondroitin-6- suifate, dermaian sulfate, keratin sulfate, etc), a cellular matrix, and/or other materials such as, but not limited to, gelatin, alginates, agar, agarose, and plant gums, and the like.

in some embodiments, the scaffold comprises, or is treated with, materials that render it non-fhrombogenic. These treatments and materia!s may aiso promote and sustain endothelial growth, migration, and extracellular matrix deposition. Examples of these materials and treatments include but are not limited to natural materials such as basement membrane proteins such as laminin and Typ IV collagen, synthetic materials such as EPTFE, and segmented po!yurethaneurea silicones, such as PURSPAN™ {The Polymer Technology Group, Inc., Berkeley, Calif.). The scaffold can also comprise anti-thrombotic agents such as heparin; the scaffolds can also be treated to alter the surface charge {e.g. , coating with plasma) prior to seeding with stem ceils. 5.14 Immortalized T-MSC and/or T-MSC-OL

Mammalian T- SC and/or T- SC-DL can be conditionally immortalized by transfection with any suitable vector containing a growth-promoting gene, thai is, a gene encoding a protein that, under appropriate conditions, promotes growth of the iransfected cell, such that the production and/or activity of the growth-promoting protein is relatabie by an external factor. In a preferred embodiment the growth-promoting gene is an oncogene such as, but not limited to, v- myc, N-myc, c~myc, p53, SV40 large T antigen, polyoma large T antigen, E1 a adenovirus or E7 protein of human papillomavirus,

Externa! regulation of the growth-promoting protein can be achieved by placing the growth-promoting gene under the control of an externaiiy-regulatable promoter, e.g., a promoter the activity of which can be con trailed by, for example, modifying the temperature of the iransfected cells or the composition of the medium in contact with the cells. In one embodiment, a tetracycline (tet)-control!ed gene expression system can be employed (see Gossen et al., Proc. Natl. Acad. Sci, USA 89:5547-5551, 1992; Hoshimaru et a!., Proc. Natl. Acad. Set. USA 93:1518- 1523, 1996), In the absence of tet, a tet-eontroiSed transacttvator (tTA) within this vector strongly activates transcription from _pilClv!V*~1, a minimal promoter from human cytomegalovirus fused to tet operator sequences. tTA is a fusion protein of th repressor (tetR) of the transposon-10- derived tet resistance operon of Escherichia coli and the acidic domain of VP 16 of herpes simplex virus. Low, non-toxic concentrations of tet (e.g., 0.01 -1.0 pg/mt) almost completely abolish transactivation by tTA.

in one embodiment, the vector further contains a gene encoding a selectable marker, e.g., a protein that confers drug resistance. The bacterial neomycin resistance gene (neo^R) is one such marker that may be employed within the present invention. Cels carrying neo^R may be selected by means known to those of ordinary skill in the art, such as the addition of, e.g., 100- 200 Mg/ml G418 to the growth medium.

Transfection can be achieved by any of a variety of means known to those of ordinary skill in the art including, but not limited to, retroviral infection, in general, a cell culture may be iransfected by incubation with a mixture of conditioned medium collected from the producer celi line fo the vector and DMEIV1/F12 containing N2 supplements. Fo example, a stem cell culture prepared as described above may be infected after, e.g., five days in vitro by incubation for about 20 hours in one volume of conditioned medium and two volumes of DMEM/F12 containing N2 supplements. Transfected ceils carrying a selectable marker may then be selected as described above.

Following transfection, cultures are passaged onto a surface that permits proliferation, e.g. , allows at least 30% of the cells to double in a 24 hour period. Preferably, the substrate is a poiyornithine/!aminin substrate, consisting of tissue culture plastic coated with polyornithine (10 g/mL) and/or lamtnin (10 Mg/mL), a polylysine/laminin substrate or a surface treated with fibronectin. Cultures ars then fed every 3-4 days with growth medium, which may or may not be supplemented with one or more proliferation-enhancing factors. Proliferation-enhancing factors may be added to the growth medium when cultures are less than 50% confluent.

The conditionally-immortalized T-MSC and/or T-MSC-DL cell lines can be passaged using standard techniques, such as by trypsinization, when 80-95% confluent. Up to approximately the twentieth passage, it is, in some embodiments, beneficial to maintain selection (by, for example, the addition of G418 fo cells containing a neomycin resistance gene). Cells may also be frozen in iiquid nitrogen for long-term storage.

Cionai ceil lines can be isolated from a conditionally-immortalized human T-MSC cell line prepared as described above, in general, such clonal ceil lines may be isolated using standard techniques, such as by limiting dilution or using cloning rings, and expanded. Clonal cell lines may generally be fed and passaged as described above.

Conditionally-immortalized human T-MSC cell Sines, which may, but need not, be clonal, may generally be induced to differentiate by suppressing the production and/or activity of the growth-promoting protein under culture conditions that facilitate differentiation. For example, if the gene encoding the growth-promoting protein is under the control of an extemaliy-regulatable promoter, the conditions, e.g., temperature or composition of medium, may be modified to suppress transcription of the growth-promoting gene. For the teiracyciine-coniroSled gene expression system discussed above, differentiation can be achieved by the addition of tetracycline to suppress transcription of the growth-promoting gene. In general, 1 g/mL tetracycline for 4-5 days is sufficient to initiate differentiation. To promote further differentiation, additional agents may be included in the growth medium. 5.15 Assays

The T-MSC and/or T-MSC-DL can be used in assays to determine the influence of culture conditions, environmental factors, molecules (e.g., biomolecules, small inorganic molecules, etc.) and the like on stem ceil proliferation, expansion, and/or differentiation, compared to T-MSC and/or T-MSC-DL not exposed to such conditions.

in a preferred embodiment, the T-MSC and/or T-MSC-DL are assayed for changes in proliferation, expansion or differentiation upon contact with a molecule. In one embodiment, for example, the invention provides a method of identifying a compound that modulates the proliferation of a plurality of T-MSC and/or T-MSC-DL, comprising contacting the plurality of T- MSC and/or T-MSC-DL with the compound under conditions that allow proliferation, wherein if the compound causes a detectable change in proliferation of the T-MSC and/or T-MSC-DL compared to a plurality of T-MSC and/or T-MSC-DL not contacted with the compound, the compound is identified as a compound that modulates proliferation of T-MSC and/or T-MSC-DL, In a specific embodiment, the compound is identified as an inhibitor of proliferation, in another specific embodiment the compound is identified as an enhancer of proliferation.

in another embodiment, the invention provides a method of identifying a compound that modulates the expansion of a piuraiity of T-MSC and/or T-MSC-DL comprising contacting the piuraiity of T-MSC and/or T-MSC-DL with the compound under conditions that aiiow expansion, wherein if the compound causes a detectable change in expansion of the piuraiity of T-MSC and/or T-MSC-DL compared to a piuraiity of T-MSC and/or T-MSC-DL not contacted with the compound, the compound is identified as a compound that modulates expansion of T-MSC and/or T-MSC-DL. in a specific embodiment, the compound is identified as an inhibitor of expansion, in another specific embodiment, the compound is identified as an enhancer of expansion.

In another embodiment, disclosed herein is a method of identifying a compound that modulates the differentiation of a T-MSC and/or T-MSC-DL, comprising contacting a T-MSC and/or T-MSC-DL with a compound under conditions that allow differentiation, wherein if the compound causes a detectable change in differentiation of the T-MSC and/o T-MSC-DL compared to a T-MSC and/or T-MSC-DL not contacted with the compound, the compound is identified as a compound that modulates proliferation of T-MSC and/or T-MSC-DL. in a specific embodiment, the compound is identified as an inhibitor of differentiation. In another specific embodiment, the compound is identified as an enhancer of differentiation.

5.16 Therapeutic Uses of Human Embryonic Stem Cell Derived Mesenchymal Stem Ceils

Mesenchymal stem ceiis derived from bone marrow (BM-MSCs) have been used as ceii based therapy for T ceil related autoimmune diseases, including multiple scierosis (MS), but due to limited sources, unstable qualify, and biosafety concerns of using ceiis derived from adult tissue, their use as a therapeutic aid has been limited.

The novel method for generating mesenchymai stem ceiis from embryonic stem cells set forth herein, and the novel T-MSC generated from this method, provid new therapies for T ceii related autoimmune disease, in particular multiple scierosis.

in certain embodiments, T-MSC given to mice pre-onset of EAE, remarkably attenuated the disease score of these animals. The decrease in score was accompanied by decreased demyelination, T ceii infiltration, and microglial responses in the centra! nervous system, as well as repressed immune ceii proliferation, and differentiation in vitro.

in certain embodiments, a graduai decline of disease score in EAE mice after treatment with T-MSC, post disease onset, was observed. In certain embodiments, T-MSC have both prophylactic and therapeutic effects on the disease.

!n certain embodiments, the immunosuppressive activity of the T-MSC account for the prophylactic effect on the disease as irradiated T-MSC, which are unlikely to replace damage myelin, and were also effective in reducing disease score, in one embodiment, irradiation does not shorten the lifespan of the T-MSC,

In certain embodiments, the therapeutic effect of the T-MSC involve immunosuppression as well as neural repair and regeneration.

In certain embodiment, EAE mice treated with T-MSC have much fewer inflammatory T cells in their central nervous system and less T ceils infiltrating the spinal cord. The T-MSC can reduce damage and symptoms caused by inflammatory T cells, making them useful in therapy and prevention of all T eel! related autoimmune diseases. T-MSC also decreased demyelination .

The characteristics of the T-SV1SC are all in marked contrast to the results obtained with bone marrow-derived mesenchymal stem cells. BM-MSCs only suppressed mouse T cell proliferation in response to anii-CD3 stimuli at low doses in vitro, and even enhanced Th1 and Th17 cell infiltration into the CNS, Autoreactive effector CD4^1' T cells have been associated with the pathogenesis of several autoimmune disorders, including multiple sclerosis, Crohn's disease, and rheumatoid arthritis. These CD4+ T ceils include Th1 and Th17 cells. There are only mild or negligible effects of human BM-MSCs on EAE mice (Gordon et al. 2008a; Zhang et a!. 2005; Payne et at 2012), A recent report showed a reduction of disease score of only 3.5 to 3.0 of EAE mice treated with human umbilical-derived MSCs (Liu et al. 2012). The results herein and those from these studies highlight the novelty and usefulness of the disclosed T-MSC.

Additionally, BM-MSC and T-MSC have very similar global transcriptional profiles, but differentia!ly express some pro- and anti-inflammatory factors. Among them, !L-6 is expressed at a much higher level in BM-MSCs than T-MSC. Moreover, IL-6 expression in BM-MSCs was double upon !FNy stimulation in vitro, whereas it remained low in the T-MSC,

iL-6 is a pleiotropic cytokine involved in crosstalk between hematopoietic/immune cells and stromal cells, including the onset and resolution of inflammation. IL-6 can promote the differentiation and functions of Th17 cells (Dong, 2008). The levels of IL-6 are elevated in mononuclear ceils in blood and in brain tissue from MS patients (Patane!la et al., 2010), as well as in serum in aged humans (Sethe et a!,, 2006). Mice lacking IL-6 receptor a at the tim of T cell priming are resistant to EAE (Leech et al., 2012), Site-specific production of IL-6 in the CNS can re-target and enhance the inflammatory response in EAE (Quintana et al., 2009), whereas !L-6- neutralizing antibody can reduce symptoms in EAE mice (Gijbeis et al., 1995). Thus, IL-6 has become a promising therapeutic target for treatment of MS.

tmmunomodulation of peripheral T cell activity and regeneration and repair of neural cells are widely recognized modes of MSC therapeutic action in MS and in EAE (A! Jumah and Abumaree, 2012; Auietta et a!., 2012; Morando et al., 2012). However, long-term functional neuronal recovery and sustained disease remission in MS needs repair of the damaged blood- brain barrier and blood-spinal cord barrier (Correale and Villa, 2007; Minagar et a!., 2012). In other words, MS is an inflammatory, neurodegenerative, and vascular disease, and effective treatment need to target al! three component.

The characteristics of T-MSC make them uniquely suited for the treatment of T eel related autoimmune diseases especially multiple sclerosis, !n particular, the T-MSC can decrease disease scores of EAE mice, but also decrease demyelination and decrease Th and Th17 proliferation, and have low expression of IL-6. These latter two characteristics make them suitable to treat other T cei! related autoimmune diseases. Additionally, the ability of the T-MSC to cross the blood-brain barrier and blood-spinal cord barrier, makes them superior as a treatment and prevention of multiple sclerosis and other autoimmune diseases related to the central nervous system.

One embodiment provided herein is a method of treating or preventing a T cell related autoimmune disease comprising the steps of administering a therapeutically effective amount of solution, cell culture or pharmaceutical preparation comprising T-MSC to the subject in need thereof. The T ceil related autoimmune diseases would include but are not limited to multiple sclerosis, inflammatory bowel disease, Crohn's disease, graft versus host disease, systemic lupus erythematosus, and rheumatoid arthritis. The subject is preferably a mammaL and most preferably human. The solution, cell culture or pharmaceutical preparation can comprise irradiated or non-irradiated T-MSC. The solution, cell culture or pharmaceutical preparation is preferably administered by injection.

Multiple sclerosis has been categorized into four subtypes; relapsing/remitting; secondary progressive; primary progressive; and progressive relapsing. The relapsing/remitting subtype is characterized by unpredictable relapses followed by long periods of remission. Secondary progressive MS often happens in individuals who start with relapsing/remitting MS and then have a progressive decline with no periods of remission. Primary progressive MS describes a small number of individuals who never have remission after their initial symptoms. Individuals with progressive relapsing, the least common subtype, have a steady neurologic decline, and suffer from acute attacks.

Provided herein is a method for treating or preventing multiple sclerosis disease in a subject in need thereof, comprising the steps of administering a therapeutically effective amount of solution, cell culture or pharmaceutical preparation comprising T-MSC as described in the preceding paragraphs, to the subject in need thereof. The multiple sclerosis can be relapsing/remitting multiple sclerosis, progressive/relapsing multiple sclerosis, primary multiple sclerosis, or secondary multiple sclerosis. The subject is preferably a mammal, and most preferably human. The solution, cell culture or pharmaceutical preparation can comprise irradiated or non-irradiated T-MSC. The solution, cell culture or pharmaceutical preparation is preferably administered by injection . Multiple sclerosis manifests in a variety of symptoms including sensory disturbance of the iimbs, optic nerve dysfunction, pyramidal tract dysfunction, bladder dysfunction, bowel dysfunction, sexual dysfunction, ataxia and diplopia attacks.

A further embodiment of the present invention is a method of treating multiple sclerosis comprising the steps of administering a therapeutica!ly effective amount of solution, cell culture or pharmaceutical preparation comprising T-MSC, to the subject in need thereof, wherein there is detectable improvement in at least one of these symptoms, at least two of these symptoms, at least four of these symptoms, at least five of these symptoms or all of these symptoms.

The Expanded Disability Status Scale (EDSS) is the most commonly used rating scale to evaluate the clinical status of patients with multiple sclerosis, it measures disability along several separate parameters: strength, sensation, brainstem functions {speech and swallowing), coordination, vision, cognition, and bowei/biadder continence. It is a well-accepted measure of disability in MS and it is not particularly difficult or time consuming to perform. The EDSS quantifies disability in eight Functional Systems (FS) and allows neurologists to assign a Functional System Score (FSS) in each of these (Kurtzke 1983),

Kurtzk defines functional systems as follows; pyramidal

cerebellar

brainstem

sensory

bowel and bladder

visua!

cerebral

other The EDSS steps 1 ,0 to 4,5 refer to people with multiple sclerosis who ar fully ambulatory. EDSS steps 5.0 to 9.5 are defined by the impairment to ambulation. The clinical meaning of each possible result is the following:

• 0,0: Normal Neurological Exam

• 1,0: No disability, minimal signs on 1 FS

· 1,5: No disability, minimal signs on 2 of 7 FS

» 2.0: Minimal disability in 1 of 7 FS

» 2,5: Minimal disability in 2 FS

• 3.0: Moderate disability in 1 FS; or mild disability in 3 - 4 FS, though fully ambulatory

· 3.5: Fully ambulatory but with moderate disability in 1 FS and mild disability in 1 or 2 FS; or moderate disability in 2 FS; or mild disability in 5 FS • 4.0: Fui!y ambulatory without aid, u and about 12 rs a day despite relatively severe disability. Able to walk without aid 500 meters

• 4.5: Fully ambulatory without aid, up and about much of day, able to work a full day, may otherwise have some limitations of full activity or require minimal assistance. Relatively severe disability. Able to waik without aid 300 meters

• 5.0: Ambuiatory without aid for about 200 meters. Disability impairs ful daily activities

• 5,5: Ambuiatory for 100 meters, disability precludes full daily activities

• 6,0: Intermittent or unilateral constant assistance (cane, crutch or brace) required to walk 100 meters with or without resting

• 6.5: Constant bilateral support (cane, crutch or braces) required to walk 20 meters without resting

• 7.0: Unable to walk beyond 5 meters even with aid, essentially restricted to wheelchair, wheels self, transfers alone; active in wheelchair about 12 hours a day

• 7.5: Unable to take more than a few steps, restricted to wheelchair, may need aid to transfer; wheels self, but may require motorized chair for full day's activities

• 8.0: Essentially restricted to bed, chair, or wheelchair, but may be out of bed much of day; retains self-care functions, generally effective use of arms

• 8.5: Essentially restricted to bed much of day, some effective use of arms, retains some self-care functions

• 9.0: Helpless bed patient, can communicate and eat

• 9,5: Unable to communicate effectively or eat swallow

• 10.0: Death due to MS

Provided herein is a method for treating multiple sclerosis disease in a subject in need thereof, comprising the steps of administering a therapeutically effective amount of solution, cell culture or pharmaceutical preparation comprising T-MSC. to the subject in need thereof wherein the subject demonstrates improvement on the Expanded Disability Status Scale of at least one point, and preferably at least two points.

There are other therapeutic agents that have been used to treat and prevent multiple sclerosis, including but not limited to, fingolimod, adrenocorticotropic hormone (ACTH), methylprednisolone, dexamethasone, !FNp-la, SFM-1 , gSiatriamer acetate, cyclophosphamide, methotrexate, a2athioprine, cladribine, cyclosporine, mitoxantrone, and sulfasalazine.

Therefore, the method of the present invention can further comprise the administration of one or more additional therapeutic agents to the subject including but not limited to, fingolimod, adrenocorticotropic hormone (ACTH), methylprednisolone, dexamethasone, ΙΡΝβ-la, tFN-1 b, gliafriamer acetate, cyclophosphamide, methotrexate, azafhioprine, cladribine, cyclosporine, mitoxantrone, and sulfasalazine. In a further embodiment these additional therapeutic agents can be administered prior to, after, or ai the same time as the T-MSC, or can be conjugated or attached to the T-MSC,

Other than T cells, T-MSC also have strong suppressive function on B cells, dendritic cells, neutrophils, N ceils, macrophage and other inflammatory and immunity related functions. Thus, T ceil, B cell, inflammatory and/or innate immunity related autoimmune diseases that can all be treated by the disclosed T-MSC include, but are not limited to, Alopecia Areata, Ank!osing Spondylitis, Anfiphospholipid Syndrome, Autoimmune Addison's Disease, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune Inner Ear Disease, Autoimmune Lymphoproliferative Syndrome (ALPS), Autoimmune Thrombocytopenic Purpura (ATP), Behcet's Disease, Bullous Pemphigoid, Cardiomyopathy, Celiac Sprue-Dermatitis, Chronic Fatigue Syndrome Immune Deficiency Syndrome (CFiDS), Chronic inflammatory DemyeSinating Polyneuropathy, Chronic Obstructive Pulmonary Disease (COPD), Cicatricial Pemphigoid, Cold Agglutinin Disease, CREST Syndrome, Crohn's Disease, Dego's Disease, Dermatomyositis, Dermatomyositis - Juvenile, Discoid Lupus, Essential Mixed Cryoglobulinemia, Fibromyalgia ~ Fibromyositis, Grave's Disease, Guiliain-Sarre, Hashimoto's Thyroiditis, idiopathic Pulmonary Fibrosis, idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, insulin Dependent Diabetes (Typ i), Type II diabetes, Juvenil Arthritis, Lupus, Meniere's Disease, Mixed connective Tissue Disease, Mulfipie Scierosis, Myasthenia Gravis, Pemphigus Vulgaris, Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, Polyglancular Syndromes, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleroderma, Sjogren's Syndrome, Stiff-Man Syndrome, Takayasu Arteritis, Temporal Arteritis/Giant Celi Arteritis, Ulcerative Colitis, Uveitis, Vasculitis, Vitiiigo.Wegener's Granulomatosis, or any acute or chronic inflammation related to burning, surgery, injury, and aiiergy.

T-MSC can be differentiated into multiple cell lineages including, but not limited to, adipocytes, myoblast cells, neural lineage cells, osteoblast cells, fibroblasts, chondrocytes, and stromal cells. These ceils derived from T-MSC (T-MSC-DL) can be used to treat multiple tissue injury, and can be used for tissue engineering, tissue repair, tissue regeneration purposes like, joint heaiing, tendon healing, connective tissue healing, neural lineage tissue and cells healing, fat tissue heaiing, bone healing, skin healing, othe wound healing, muscle healing, cartilage heaiing, smooth muscle healing, myocardiac heaiing, epitheiia tissue heaiing, ligament heaiing, stroma repair, etc.

Specifically, T-MSC can be differentiated into neural iineage cells, which can be used to treat many neural disease including but not limited to Agraphia, Alzheimer's disease,

Amyotrophic lateral sclerosis, Aphasia, Apraxia, Arachnoiditis, Ataxia Telangiectasia, Attention deficit hyperactivity disorder, Auditory processing disorder, Autism, Alcoholism, Asperger's syndrome, Bipolar disorder, Bell's palsy, Brachial plexus injury, Brain damage, Brain injury, Brain tumor, Canavan disease, Capgras, Causalgia, Centra! pain syndrome, Centra! pontine

myelolysis, Centronuclea myopathy, Cephalic disorder, Cerebral aneurysm, Cerebral arteriosclerosis, Cerebral atrophy, Cerebrai gigantism, Cerebrai palsy, Cerebral vasculitis, Cervical spinal stenosis, Charcot-Marie-Tooth disease, Chiari malformation, Chorea, Chronic fatigue syndrome, Chronic inflammatory demye!inating polyneuropathy (CIDP), Chronic pain, Coffsn-Lowry syndrome. Coma, Complex regional pain syndrome, Compression neuropathy, Congenital facial diplegia, orttcobasal degeneration, Crania! arteritis, Craniosynostosis,

CreutzfeSdt-Jakob disease, Cumulative trauma disorders, Cushing's syndrome, Cytomegalic inclusion body disease (CI8D), Cytomegalovirus Infection, Dandy-Walker syndrome, Dawson disease, De Morsier's syndrome, Dejerine-Klumpke palsy, Dejerine-Sottas disease, Delayed sleep phase syndrome, Dementia, Dermatomyositis, Developmental dyspraxia, Diabetic neuropathy, Diffuse sclerosis, Downs syndrome, Dravet syndrome, Dysautonomia, Dysca!cu!ia, Dysgraphia, Dyslexia, Dystonia, Empty sella syndrome, Encephalitis, Encephalocele,

EncephaSotrigeminal angiomatosis, Encopresis, Epilepsy, Erb's palsy, Erythromelaigia, Essentia! tremor, Fabry's disease, Fahr's syndrome, Fainting, Familial spastic paralysis, Febrile seizures, Fisher syndrome, Friedreich's ataxia, Fibromyalgia, Foville's syndrome, Fetal Alcohol Effect, Gaucher's disease, Gerstmamfs syndrome, Giant cell arteritis, Giant cell inclusion disease, G!oboid Cell Leukodystrophy, Gray matter heterotopia, Gui!lain-Barre syndrome, HTLV-1 associated myelopathy, MaSlervorden-Spatz disease, Head injury, Headache, Hemifacial Spasm, Hereditary Spastic Paraplegia, Heredopathia atactica polyneuritiformis, Herpes 20Ster oticus, Herpes zoster, Hirayama syndrome, Ho!oprosencephaly, Huntington's disease,

Hydranencepha!y, Hydrocephalus, Hypercortisotism, Hypoxia, Immune-Mediated

encephalomyelitis, Inclusion body myositis, Incontinentia pigmenti. Infantile phytanic acid storage disease, infantile Refsum disease, Infantile spasms, Inflammatory myopathy, intracranial cyst, Intracranial hypertension, Joubert syndrome, arak syndrome, Kearns-Sayre syndrome, Kennedy disease, Kinsbourne syndrome, lippel Foil syndrome, Krabbe disease, Kugeiberg- We!ander disease, Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Klefmer syndrome, Lateral medullary (Wallenberg) syndrome, Learning disabilities, Leigh's disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, Leukodystrophy, Lewy body dementia, Lissencephaly, Locked-ln syndrome, Lou Gehrig's disease {See amyotrophic lateral sclerosis), Lumbar disc disease, Lumbar spinal stenosis, Lyme disease - Neurological Sequelae, Machado- Joseph disease (Spinocerebe!iar ataxia type 3), Micrencephaly, Macropsia, Megalencepha!y, Melkersson-Rosenthal syndrome, Menieres disease, Meningitis, Menkes disease, Metachromatic leukodystrophy, Microcephaly, Micropsia, Migraine, Miiler Fisher syndrome, Mini-stroke (transient ischemic attack), Misophonia, Mitochondria! myopathy, Mobius syndrome, Monomelic amyotrophy, Motor Neurone Disease - see amyotrophic lateral sclerosis, Motor skills disorder, Moyamoya disease, Mucopolysaccharidoses, Muitt-infarct dementia, Multifocal motor neuropathy, Multiple sclerosis, Multiple system atrophy, Muscular dystrophy, Mya!gic

encephalomyelitis, Myasthenia gravis, Myelinociastic diffuse sclerosis, Myoclonic

Encephalopathy of infants, Myoclonus, Myopathy, Myotubular myopathy, Myotonia congenita, Narcolepsy, Neurofibromatosis, Neuroleptic malignant syndrome, Neurological manifestations of AIDS, Neurological sequelae of lupus, Neuromyotonia, Neuronal ceroid lipofuscinosis, Neuronal migration disorders. Neurosis, Niemann-Pick disease, Non 24-hour sleep-wake syndrome, Nonverbal learning disorder, Neurological disorder, O'Suiiivan-ycLeod syndrome, Occipital Neuralgia, Occult Spinal Dysraphism Sequence, Ohtahara syndrome, Olivopontocerebellar atrophy, Opsodonus myoclonus syndrome, Optic neuritis, Orthostatic Hypotension, Otosclerosis, Overuse syndrome, Palinopsta, Paresthesia, Parkinson's disease, Paramyotonia Congenita, Paraneoplastic diseases, Paroxysmai attacks, Parry-Romberg syndrome, Pelizaeus-Merzbacher disease, Periodic Paralyses, Peripheral neuropathy, Pervasive developmental disorders, Photic sneeze reflex, Phytanic acid storage disease, Pick's disease, Pinched nerve, Pituitary tumors, PMG, Polyneuropathy, Polio, Polymicrogyria, Polymyositis, Porencephaly, Post-Polio syndrome, Postherpetic Neuralgia (PHN), Postural Hypotension, Prader-Willi syndrome, Primary Lateral Sclerosis, Prion diseases, Progressive hemifacial atrophy, Progressive multifocal

leukoencephalopafhy, Progressive Supranuclear Palsy, Pseudotumor cerebri, Quadriplegia, Rabies, Ramsay Hunt syndrome type I, Ramsay Hunt syndrome type il, Ramsay Hunt syndrome type III - see Ramsay-Hunt syndrome, Rasmussen's encephalitis, Reflex neurovascular dystrophy, Refsum disease, Repetitive stress injury, Restless legs syndrome, Retrovirus- associated myelopathy, Rett syndrome, Reye's syndrome, Rhythmic Movement Disorder, Romberg syndrome, Saint Vitus dance, Sandhoff disease, Schilder's diseasefdisarnbiguation needed], SchizencephaSy, Sensory integration dysfunction, Septo-optic dysplasia, Shaken baby syndrome, Shingles, Shy-Drager syndrome, Sjogren's syndrome, Slee apnea, Sleeping sickness, Snatiation, Sotos syndrome, Spasticity, Spina bifida, Spinal cord injury, Spinal cord tumors, Spinal muscular atrophy, Spinocerebellar ataxia, Split-brain, Steele-Richardson- Olszewski syndrome, Stiff-person syndrome, Stroke, Sturge-Weber syndrome, Subacute sclerosing panencephalitis, Subcortical arteriosclerotic encephalopathy, Superficial siderosis, Sydenham's chorea, Syncope, Synesthesia, Syringomyelia, Tarsal tunnel syndrome, Tardive dyskinesia, Tardive dysphrenia, Tarlov cyst, Tay-Sachs disease, Temporal arteritis, Tetanus, Tethered spinal cord syndrome, Thomsen disease, Thoracic outlet syndrome, Tic Douloureux, Todd's paralysis, Tourette syndrome, Toxic encephalopathy, Transient ischemic attack,

Transmissible spongiform encephalopathies. Transverse myelitis, Traumatic brain injury, Tremor, Trigeminal neuralgia, Tropical spastic paraparesis, Trypanosomiasis, Tuberous sclerosis, Ubisiosis, Template:Un!polar depression, Von Hippel-Lindau disease (VHL), Viliuisk Encephalomyelitis (VE), Wallenberg's syndrome, Werdnig-Hoffman disease, West syndrome, Whiplash, Williams syndrome, Wilson's disease.

5J7 Uses of T-MSC as Delivery Systems

Because it has been shown that the T-MSC of the present invention have the unique ability to cross the blood-brain barrier and the blood-spinal cord barsier, a further embodiment of the present invention is a method of using T-MSC for delivery of agents through the blood brain barrier and/or the blood spinal cord barrier, by attaching or conjugating the agent to the T-MSC to form a complex; and administering the T-MSC-agent complex to a subject, wherein the T-MSC cross the blood- brain and/or the blood-spinal cord barrier and deliver the agent to the central nervous system. The T-MSC may be in the form of a single cell, a cell culture, a solution or a pharmaceutical preparation. Agents would include but are not limited to chemicals, drugs, proteins, DNA, RNA, antibodies, and small moiecuies.

A further embodiment of the present invention is a delivery system for the delivery of agents through the biood brain barrier and/or the blood spina! cord barrie comprising T-MSC and an agent conjugated or attached to the T-MSC.

The ability to permeate the blood-brain barrie and the blood-spinal cord barrier would be useful in the treatment and prevention of diseases including but not limited to neurological disorders, multiple sclerosis, cancer, Parkinson's Disease, Alzheimer's Disease, Huntington's Disease, meningitis, encephalitis, rabies, epilepsy, dementia, Lyme^'s Disease, stroke, and amyotrophic lateral sclerosis, as well as brain and spina! cord injury. Thus, a subject in need thereof would have a disease or be at risk for a disease in which the blood- brain barrier and/or blood-spinal cord barrier is involved. Thus, a further embodiment of the present invention is a method of treating a disease or injury, by attaching or conjugating an agent to the T-MSC to form a complex; and administering the T~MSC~agent complex to a subject in need thereof, wherein the T-MSC cross the blood- brain and/or the blood-spinal cord barrier and deliver the agent to the central nervous system, and the agent is used as a treatment or prevention of the disease or injury of the subject. Since the T-MSC have strong migration ability and infiltration ability, it can also been used as carrier for tumor/cancer therapy to carry anti-tumor drugs and proteins. The T- MSC may be in the form of a single cell, a cell culture, a solution or a pharmaceutical preparation. Agents include, but are not limited to, chemicals, drugs, proteins, DNA, RNA, micro- RNA, non-coding RNA, antibodies, small molecules and/or nano particles.

Agents that are useful in the treatment and prevention of diseases include, but ARE not limited to, antibiotics, anti-viral agents, anti-fungal agents, steroids, chemotherapeutics, anti- inflammatories, cytokines, and/or synthetic peptides.

Proteins and peptides would also be useful to conjugate to the T-MSC and would include erythropoietin (EPO), anti-beta-amyloid peptides, tissue plasminogen activator (TPA), granulocyte colony stimulating factor (G-CSF), interferon (IFN), growth factor/hormone, anti- VEGF peptides, anti-TNF peptides, NGF, HGF, IL-2, CX3CL1 , GOV, CPT-11 , cytosine deaminase, HSV-TK, carboxyesterase, oncolytic virus, TSP-1 , TRAIL, FASL IL-10, and TGFb. Proteins and peptides that bind to particular receptors and block these receptors would also be useful and are contemplated by the current invention to be attached to the T-MSCs.

DNA and RNA tha coded for therapeutic proteins and peptide would a!so be useful to conjugate to the T- SC for delivery across the biood- brain barrier and/or the b!ood-spinai cord barrier.

The terms "antibody" and "antibodies" include polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab')₂ fragments. Polyclonal antibodies are heterogeneous populations of antibody molecules that are specific for a particular antigen, while monoclonal antibodies are homogeneous populations of antibodies to a particular epitope contained within an antigen. Monoclonal antibodies are particularly useful in the present invention.

Any agent that would block the activation, expression and/or action of a molecule or the receptor of the molecuie in the pathway related to any disease in which crossing the blood-brain barrier and/or blood-spinal cord barrier is useful could be attached or conjgaied to the T-MSCs. Such agents include but are not limited to chemicals, phytochemica!s, pharmaceuticals, biologies, small organic molecules, antibodies, nucleic acids, peptides, and proteins,

inhibiting a pathway can also be effected using "decoy" molecules which mimic the region of a target molecui in the pathway binds and activates. The activating molecule would bind to the decoy instead of the target, and activation could not occur.

inhibition can also be effected by the use of a "dominant!y interfering" molecule, or one in which the binding portion of activating molecule is retained but the molecule is truncated so that the activating domain is lacking. These molecules would bind to receptors in the pathway but be unproductive and block the receptors from binding to the activating molecule. Such decoy molecules and dominaniiy interfering molecuie can be manufactured by methods known in the art, and attached or conjugated to the T-IV1SG for delivery across the blood-brain or blood -spinal cord barrier.

A method for delivery of agents across the blood-brain and/or blood-spinal cord barrier is also useful for diagnostic agents, including but not limited to chemicals, antibodies, peptides, proteins, DNA, and RNA. Such agents in order to be usefui for diagnosis must have a means of being visualized and/or quantified. Such means include, but are not limited to, fluorescence, biomarkers, dyes, radioactive isotypes labels and/or nanoparticles.

Such a method for delivery and a delivery system would be useful for the diagnosis of neurological disorders, multiple sclerosis, cancer, Parkinson's Disease, Alzheimer's Disease, Huntington's Disease, meningitis, encephalitis, rabies, epilepsy, dementia, Lyme's Disease, stroke, and amyotrophic lateral sclerosis, as weli as brain and spsnai cord injury, Thus, a further embodiment of the present invention is a method of diagnosing a disease or injury, by attaching or conjugating the agent to the T-MSC to form a complex; and administering the T- SC-agent complex to a subject in which a disease is suspected, wherein the T-MSC cross the biood- brain and/or the biood-spirtal cord barrier and deliver the agent to the centra! nervous system. The T- MSC may be in the form of a single ceil, a ceil culture, a solution or a pharmaceutical preparation- Agents would include but are not limited to chemicals, drugs, proteins, DNA, RNA, antibodies, and small molecules.

Agents, no matter the type and whether for treatment, prevention, or diagnosis, can be conjugated or attached to the T-MSC by any method known in the art including, but not limited to, synthetic extracellular matrix, aiginate-poiy-L-Lysine encapsulate and/or container.

In certain embodiments, large scale production at industrial level of manufacturing is included in the present disclosure, methods of which are well known in the art. In certain embodiments, the large scale production includes the use of a Hyper-STACK 2D culture system and/or a Mierocarrier 3D bioreactor.

6. EXAMPLES

Example 1. Derivation of T-MSC

Material and Methods

The following reagents and materials were obtained from the below-described sources;

Customed mTeSRI Medium; Stem Cell Technology, inc.

BMP4: Stemgent or other vendors

SB431542: Cayman Chemical or other vendors

A83-01 : Stemgent or other vendors,

ALK5 inhibitor; Stemgent or other Vendors

DMEM/F12: GIBCO Life Technologies

alpha-MEM: GI8CO Life Technologies

Fetal Bovine Serum; GIBCO Life Technoiogies or other vendors

CT2 hESC line derived at the University of Connecticut Stem Ceil Core was cultured for two passages on irradiated mouse embryonic fibroblast (MEF) as feeders. The hESCs were then split on plates coated with Matrigel (BD Biosciences, San Jose, CA) and cultured in mTeSRI {Ludwig et al., 2006) (Stem Ceil Technoiogies, Vancouver, Canada). ES!-017, ES!-051 , ESi-053, ES!-049, and ESI-35 human embryonic stem cells were purchased from BioTime, Inc. (CA).

Derivation of T-MSC

As shown in FIG. 1 , hESCs at -80% conf!uency on the Matrigel-coated plates were digested with Dispase at 1 mg/mS for 5-10 min. The cel!s were then washed with mTESRI medium once and split as small clumps or single cel!s onto Matrigel-coated plate and cultured in mTeSRI for 12 hr, Then the culture medium was replaced by a trophobiast-formation medium containing BMP4 (2-100 ng/mi), or optional A83-01 {0.1 -1 μΜ). After culture for 48-72 hr, the ceiis changed from hESC-!ike morphology into trophoblasi-like morphology featured by flat, enlarged cell size, small nucSear/cytosol ratio, and diffuse cell borders. The ceils were digested with Tryp- IE and washed with MSC growth medium (alpha-MEM containing 20% fetal bovine serum and non-essential amino acids). The cells were then plated onto Matrigei-coated plates at a density of 5, GOG ce!is/cm² The medium was changed after 24 hr, and then changed every 3-4 days. After 6 more days, the cells were differentiated into spindle-like cells similar to the morphology of typical MSCs. Morphology of Day2 Trophab!ast are shown in FIG. 2A, morphology of Day 5 pre-T-MSC are shown in FiG. 2B, morphology of T-MSC are shown in FIG, 2C,

Derivation of HB-MSC

CT2 hESC cells were differentiated into EB cells and then enriched for H8 as previously described (Lu et ai., 2008); Lu et al._« 2007)). 50-80% confluent hEC cell on the Matrigel p!ate were digested with Dispase (1 mg/mi for 5 to 10 minutes) and then washed with EB formation basal medium, HPGM (Lonza, Waiksvi!le, Maryland), or STEMLINE l/l I Hematopoietic Ste!i Cell Expansion Medium (Sigma, St. Louis, Missouri), or StemSpan H3000 (Stem Cell Technologies, Vancouver, Canada), or IMDM with 10% FBS, or DMEDM/F12 with 10% FBS. Cells were then cultured in EB formation medium supplemented with 50 ng/ml of VEGF (Peprotech) and 50 ng/mi of BMP4 (Stemgent) for 48 hours on ultra-low plate at a density of about 2-3 million cells/ml. After 48 hours, half the culture medium was replaced with fres EB formation medium plus 25-50 ng/mi of bFGF.

Four days later, EB ceiis formed in the medium were harvested and dissociated into single cells with TrypLE (!nvitrogen) at 37"C for 2-3 minutes. Cells were washed and resuspended at 1-5 million cells/ml in EB formation basal medium. The single cell suspension was then mixed at 1 ;10 with Hemangioblast Growth Medium (Stem Ceil Technologies, Vancouver, Canada).

Blast ceil growth medium (BGM) were made as follows: To 100 mi Serum-free methylceliulose CFU medium (Stem Cell Technologies, H4436 or H4536), added with VEGF, TPO and FLT3-Ligand to 50 ng/ml, bFGF to 20-50ng/ml, 1 ml of EX-CYTE Growth Enhancement Media Supplement and 1 ml of Pen/Strap, mix well.

The mixtures were vortexed and plated onto ultralow plates by passing; through a 18G needle and cultured for 5-9 days at S/^C with 5% C0₂.

Singie ce!is were then re-suspended in MSC medium containing: 1 ) 10-20% FBS in alpha-MEM (!nvitrogen) or 2) 10-20% KOSR alpha-MEM, 3) 10-20% FBS DMEM high-glucose, or 4) 10-20% KOSR DMEM high-glucose, and cultured on either Matrigel, gelatin, vitronectin, !aminin, fibronectin, or collagen I coated plates at a density of 00-5,000 cell/cm². The medium was changed after 24 hours and refreshed every 2-4 days. After 8-12 days the celis graduai!y differentiated into spindle-like ceils similar to typical MSCs.

Derivation of MSG .through SB431542

This method was published previously (Chen et at, 2012).

Results

!t was found that the method generated T-MSC that have superior efficiency, yield and purity. As shown in the bottom panel of FIG. 1 , on Day 10, T-MSC already generated >90% purify of MSG with 10 fold cell number increase, whereas other methods either did not have any MSG or only had ver low purity of MSCs. On Day20, T-MSC already had 3000 fold expansion with >99% purity of MSCs, whereas the other methods on y expanded 20 fold at most. By day 30, 0.1 million of hESC generated 50 billion of T-MSC, that is a 500,000 fold expansion of the original hESCs, whereas the other methods only expanded 3000 fold at most.

Example 2. Characterization of T-MSC cells

The T-MSC cells obtained in Example 1 were further analyzed using fiow cytometry immunofluorescence staining .

Materials and Methods

Fiow cytometry staining was used to characterize the T-MSCs, Ceils were washed and blocked with 2% BSA in PBS, and stained with antibodies for various cell surface markers Trop~2 {Trp-2, ©Bioscience), CD31 , CD34, CD29, CD73, CD90, CD105, CD44, CD45, CD 146, GDI 66, HIA-ABC, HLA-DR, MLA-G (BD Bioscience or eBioscience) by following the manufacturers' instructions. Data were collected on FAGS LSR Si Flow Cytometer using FACSDiva software (BD Bioscience). Post-acquisition analysis was performed with the FiowJo software (Treestar).

Resuits

The attached ceils obtained from Day 2 irophobiast, Day 5 pre-T-MSC and Day 9 T-MSC were stained with CD73 and Trop-2. The trophobiast cells only expressed high levels of Trop-2 {greater than 95%), but iess than 1 % of CD73 {FIG, 3A); the pre-T-MSC at day 5 has more than 50% of ceils express both Trop-2 and CD73, 40% of the cells express only CD73 (FIG. 3B); T- MSC at day 9 of hESC differentiation has iess than 1% of the ceils express Trop-2, and 99% of ceils express oniy CD73 (FIG. 3C).

Further characterization of the T-MSC by FAGS staining of multiple ceil surface markers show T-MSC express <3% of Trop-2, <1% of CD31 , CD34. >99% of CD73, >95% of CD90, >90% of CD105, >99% of CD44 and >80% of CD29 (FIGS. 4 A-H). Example 3. T-MSCs have a Stronger Inhibition on T Ceil Functions In Vitro than B -MSC hEs-MSCs and SM-MSCs were compared for their ability to inhibit T cell proliferation in vitro foliowing antigen stimulation. Material and Methods

Culture of BM-MSCs

B -MSCs were derived from BM mononuclear ceils (BSVI NCs) or obtained from AilCeils, inc. (Alameda) and Lonza (Basel, Switzerland) BM NCs. For derivation, BMMNCs were thawed and plated onto tissue culture plastic dishes in uMEM + 20% FBS. Adherent celis began to appear within the first 4-5 days and fed every 3 days untii day -10-12, when celis were harvested and replated at 3.000-5,000 ce!!s/cm².

The in vitro assay for T eel! proliferation was performed using lymphocytes isolated from mouse peripheral lymph nodes. These lymphocytes were labeled with 5 μΜ of carboxyfiuorescein succinimidyi ester (CFSE) to track their proliferation by monitoring CFSE dilution in their daughter ceils, for 10 minutes at 37 °C. 10,000 T- SCs or BM-MSCs were mixed with 100,000 lymphocytes per well in a 96-weli plate, and the cells were stimulated for proliferation with plate-bound anti-CD3 {at 0.3, 1 pg/mi) and soluble anti-CD28 antibodies {1 pg/ml, eBioscience, CA). The cells were collected 3 days after the stimulation, followed by FACS staining with anti-CD4 and anti-CDS antibodies (BD Bioscience, CA). CFSE dilution was gated on CD4+ and CD8+ T cells, respectively.

Resuits

Using the in vitro assay with mouse lymphocytes, it was found T-MSCs inhibited the proliferation of mouse CD4+ and CD8+ T cells when stimuiated with anti-CD3 antibody at 0,3 and 1 pg/mi, whereas BM-fvlSC only did so when the T cells were stimulated with anti-CD3 antibody at low doses, i.e., 0.3 pg/ml (FIG. 5)

Example 4. T-MSCs Attenuate the Disease Score of EAE Mice Because it has been shown that BM-MSCs can attenuate the disease progression of the mouse rnodeS of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), the T- SCs obtained in Example 1 wer injected into mic with EAE to determine if they would have ihe same effect.

Materiais and Methods

Derivation of MSC through SB431542 : the MSC derived from this method will be called hES-M5C(SB}, This Method was published previously (Chen et ai., 2012).

The mouse EAE modei was induced as previousiy described (Stromnes and Goverman, 2006). C57BL/6 mice were subcutaneously injected with a mixture of myelin oligodendrocyte glycoprotein peptide 35-55 (MOG^{35 5~}), Freund^'s adjuvant, and pertussis toxin contained in the EAE induction Kit {Hooke Laboratories, Inc, MA. (Cat. # EK-0114)) following the manufacturers protocol and as described in Ge et al. {2012). BM-MSC, T-MSC or hES-MSC(SB) at 10⁶ cells/mouse or PBS (a vehicle control) was iniraperftoneal (i.p.) injected on day 6 (for pre-onset) or 18 (for post-onset) after the immunization. The disease score was monitored on the mice every day for up to 31 days.

The disease scoring system is as follows:

0; no sign of disease;

1 : loss of tone in the tali;

2: pariiai hind limb paraiysis:

3: complete hind Simb paralysis;

4: front iimb paralysis; and

5: moribund

{Stromnes and Goverman, 2006).

Results

As shown in F!G. 6, the T-MSCs significantly attenuated the daily disease scores when injected at 6 days or pre-onset of disease, showing a prophylactic effect of the T- SCs. Mice injected with BM-MSC did not attenuate the disease score, hES-MSC(SB) had a partial effect in attenuating the disease score but not as good as T-MSC.

Example 5, Multi-lineage differentiation of T-MSC

aterials and Methods

Osteogenesis , chondrogenesis and adipogen esis of T-MSC

STEMPRO Osteogenesis and Chondrogenesis Differentiation Kits (!nvitrogen, Grand Island, NY) were used for osteogenesis and chondrogenesis, and the Hycione AdvanceSTEM Adipogentc Differentiation kit (Thermo Scientific, Logan, UT) for adipogenesss, following the manufacturers' instructions.

Results

As shown in FiG. 7. T- SC had good potency in differentiating into all the 3 lineages of mesoderm tissues, osteoblasts, chondrocyte and adipocytes. Thus, T-MSC can be used as source for tissue regeneration, tissue engineering and tissue repair, Example 6. T-MSC are different from hES-HB-MSC and B&8-MSC

Microarray analysis was performed to compare the gene expression profile of T-MSC, hES-HB-MSC and BM-MSCs.

Materials and Methods

For microarray analysis, RNA of hES~M.SC at passages 2-4 or BM-MSC at passage 3 were harvested with Trizol (Invitrogert, CA) following the manufacturer's protocol. The HumanHT- 12 v4 Expression SeadChip (l!iumina, San Diego, CA) was used to analyze the gene expression profile of the ce!is. Data were analyzed using Genome Studio V2011.1. Two BM-MSC cell iines from different sources were used, and two hES-MSC ceii lines, derived from H9 and SV1A09, were used.

Results

As shown in FIG, 8, the overall expressional profiles of some key cytokines, transcription factors, ceil surface markers are very different between these 3 different ivlSCs. T-MSC may piay different roles in immunosuppression and tissue regeneration.

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While the disclosure has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for the elements thereof without departing from the scope of the disclosure. In addition, many modifications may be made to adapt the teaching to particular use, application, manufacturing conditions, use conditions, composition, medium, size, and/or materials without departing from the essential scope and spirit of the disciosure. Therefore, it is intended that the disclosure not be limited to the particular embodiments and best mode contemplated for carrying out as described herein. Such modifications are intended to fall within the scope of the appended claims.

All references cited herein are incorporated by reference in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Claims

CLAIMS:

1. A method of producing; trophobiast-derived mesenchymal stem cells (T- MSCs) from human embryonic stem eels (hESCs) or induced piuripotent stem celis (iPSCs), the method comprising:

(a) cuituring hESCs or iPSCs in a medium comprising a bone morphogenetic protein (BMP) for a suitable first time period sufficient for the hESC or iPSC to differentiate into trophoblasts, or together with a TGFb inhibitor to increase the differentiation efficiency;

(b) dissociating trophoblast celis into single cells;

(c) re-piafsng the trophob!ast cells onto gelatin, vitronectin, !aminin, fibronectin, Matrigel or coSlagen-coated plates; and

(d) cuituring the trophoblast ceils for a suitable second time period in a mesenchymal stem cell (MSC) growth medium containing L!F, bFGF or PDGF to increase expansion efficiency

2. A method of producing T-MSCs from hESCs or iPSCs, the method comprising:

(a) cuituring hESCs or IPSCs in a medium comprising BIVIP4 for 2-5 days for the hESC or iPSC to differentiate into trophoblasfs;

(b) isolating the trophoblasts;

(c) dissociating trophoblast cells from the isolated trophoblasts;

(d) re-plating the trophobiast celis onto atrigei-coated plates; and

(e) cuituring the trophoblast celis for 4-10 days in a MSC growth medium containing serum, KOSR or other serum-fre medium in an amount sufficient to induce differentiation of the single celis into mesenchymal stem cells; wherein at least about 90% of the mesenchymal stem celis express CD73,

3. The method of Claim 1 wherein the first time period is about 1 ½ days to about 4 days.

4. The method of Claim 3 wherein the first time period is about 2 days to about 5 days,

5. The method of Claim 1 wherein the second time period is about 4 days to about 10 days.

6. The method of Claim 5 wherein the second time period is about 6 days to about 8 days.

7. The method of CSaim 2, wherein the medium further comprises a ΤΘΡβ inhibitor to increase the differentiation efficiency.

SO

8. The method of Claim 7, where the TGFp inhibitor is an SB431542, A83- 01 or ALK5 inhibitor.

9. The method of Claim 1 and 2 wherein, prior to the first step of Claim 1 , hESCs are cultured comprising the following steps;

0) cuituring to about 80% confiuency on Matrigei-coaied plates;

(is) dissociating under suitable conditions;

(iii) isolating; and

(iv) washing.

10. The method of Claim 1 , wherein the BMP is EMP4, BMP2, BMP/, growth and differentiation factor-5 (GDFS) or a combination thereof.

1 1 . The method of C!aim 10, wherein the concentration of BMP4 is about 1 to about 100 ng/ml, the concentration of BMP2 is about 100 to about 500 ng/mi, the concentration of BMP7 is about 100 to about 500 ng/mi, and the concentration of growth and differentiation factor-5 (GDFS) is about 10 to about 50 ng/ml.

12. The method of Claim 1 , wherein the BMP is a factor or a peptide that is functionaiiy and biologically equivalent to BMP4 with respect to differentiation of MSGs from hESCs, derivatives of BMP4, formulations of BMP4, N-terminal peptide portions of 8MP4, C~terminai portions of BIV5P4, or a combination thereof,

13. The method of Ciaim 2, wherein the concentration of 8MP4 is about 2 ng/ml to about 100 ng/mi.

14. The method of Ciaim 2, wherein the concentration of BMP4 is about 1 ng/ml to about 10 ng/mi.

15. The method of Ciaim 8, wherein the concentration of AS3-01 is about 0.1 μΜ to about 5 Μ

16. The method of Ciaim 8, wherein the concentration of S843 542 is about 1 μΜ to about 20μΜ

17. The method of Claim 1 to 16, wherein the produced T-MSC are CD73\ CD105\ CD90⁺, and have a purity at ieast about 90%.

18. Trophobiast-derived MSCs (T-MSC) produced by the me hod of Claim 1 or Claim 2.

19. The methods of Ciaim 1 or Claim 2, wherein the hESCs are replaced with embryonic stem ceiis (ESCs) from a non-human mammaiian source, and non-human mammaiian MSCs are produced.

20. The methods of Ciaim 1 or Ciaim 2, wherein the hESCs are replaced with induced piuripotent stem ceils (iPSCs), and iPSC derived T-MSCs are produced.

21 . Adipocytes, chondrocytes, osteoblast cells, neural lineage ceiis, myoblast ceils, stroma! ceils and fibroblast cells generated from the T-MSCs of Claim 18.

SI

22. A population of trophobiast-derived MSCs (T-MSCs) produced by the method of Claim 1.

23. The population of Claim 22, wherein the T-MSC are CD73\ CD105*. and CD90⁺.

24. A method of treating an autoimmun disease or condition in a subject, the method comprising administering to a subject a suitable dose by a suitable administration route of T-MSCs produced by the methods of Claim 1 to 17.

25. A method of immunomodulation in a subject, the method comprising administering to a subject a suitable dose by a suitable administration route of T-MSCs produced by the methods of Claim 1 to 17 and 19 to 20.

26. A method for prevention or inhibition of immunorejection in a subject during tissue or organ transplantation, the method comprising administering to a subject a suitable dose by a suitable administration route of T-MSCs produced by the method of Claims 1 to 17 and 19 to 20.

27. The cell population of Claim 22, wherein the cells are at least about 90%

CD73+.

28. The ceil population of Claim 22, wherein the ceils are at least about 95%

CD73+.

29. The cell population of Claim 27 or Claim 28, wherein the cells are CD73 (>98%>, CD90 f>95%), CD105 (>90%), CD44 <>95%), CD29 (>80%).

30. The cell population of Claims 27 to 29, wherein the cells are negative for the endothelial marker CD31 and hematopoiesis marker CD34.

31 . A method of selecting clinical grade trophobiast-derived mesenchymal stem cells (T-MSCs) for treating autoimmune diseases, comprising selecting T-MSCs having the following characteristics; (\) contain >95% of eels expressing group- 1 markers; (ii) contain >80% of ceils expressing group 2 markers; (iii) contain <5% of ceils expressing group-3 markers; (iv) express 11-10 and TGFp; (v) contain <2% of cells expressing 11-6, 11-12 and TNFa; and (vi) contains <0.001% of cells co-expressing all group-4 markers, wherein group- 1 markers are CD73, CD90, CD105, CD 146, CD166, and CD44, group-2 markers are CD13. CD29, CD54, CD49E, group-3 markers are CD45, CD34, CD31 and SSEA4, and group-4 markers are OCT4, NANOG, TRA-1 -60 and SSEA4.

32. The method of Claim 31 , wherein the cells do not express IL-6, SL12 and

TNFa.

33. The method of Claim 31 , wherein the cells express TGF-p1 , TGF-fJ2 and

IL10.

S2

34. The method of Claim 31 , wherein the ceils do not express MMP2 and

RAGE.

35. The method of Claim 31 , wherein the T-MSC eels have Sow expression of iFWyRI and !FNyR2 compared to expression of IFNyRl and IFNyR2 by bone marrow- derived mesenchymal stem cells (BM-MSC).

36. A method of producing a population of modified T-MSCs, comprising modifying mesenchymal stem ce!is (MSC) to produce modified T-MSCs having the following characteristics: (i) express !L-10 and TGFp; and (it) contain <2% of cells expressing !L-8, !L-12 and TNFa.

37. The method of Claim 36, further comprising modifying the MSC fo decrease the expression of il~6₍ lL-6 receptor, IL12, TNFa, or a combination thereof.

38. The method of Claim 36, further comprising modifying the MSC to decrease the expression of MP2, RAGE or combination thereof.

39. The method of Claim 36, further comprising modifying the MSC to increase the expression of TGF-βΙ , TGF-f32 and/or IL10 through sh NA or miRNA.

40. The method of Claim 36, further comprising modifying the MSC to decrease the expression level of !FNyRI , !FNyR2, IFNy, or a combination thereof.

41 . The method of Claim 1 or Claim 2, further comprising a step of irradiating the TSMCs.

42. The method of Claim 1 or Claim 2, wherein the method produces 1 x 10⁶ to 5 x 10^1ϋ T-MSCs or produces 1 x 10^s human embryonic stem cells or induced piuripotent stem cells,

43. The method of Claim 31 , wherein the characteristics of the selected T- MSCs further comprise the characteristics of expressing CD73 and of low expression or non-expression of il-6,

44. The method of Claim 31 , wherein the characteristics of the selected T- SCs further comprise the characteristics of: expressing at least one cell marker from CD90, CD1G5, CD13, CD29, CD54, CD146, CD166 and CD44; low expression or non- expression of at least one ceil marker chosen from CD34, CD31 , and CD45; and iow expression or non-expression of at least one marker from MMP, RAGE, IFNyR , !FNyR2, lL-12, TNFa and VCAML

45. The method of Claim 43, wherein the selected T-MSCs are further subjected to irradiation.

46. The method of Claim 44, wherein the selected T-MSCs are further subjected to irradiation.

47. A cell culture comprising; the selected T-MSCs of Claim 43.

48. A cell culture comprising the selected T-MSCs of Claim 44.

S3

49. A ceil culture comprising the selected T-MSCs of Claim 45.

50. A ceil culture comprising the selected T-MSCs of Claim 46.

51 . A pharmaceutical preparation comprising the selected T-MSCs of Ciaim

43 and a pharmaceutically acceptable carrier.

52. A pharmaceuticai preparation comprising the selected T-MSCs of Ciaim

44 and a pharmaceutically acceptable carrier.

53. A pharmaceutical preparation comprising the selected T-MSCs of Ciaim

45 and a pharmaceutically acceptable carrier.

54. A pharmaceutical preparation comprising the selected T-MSCs of Ciaim

46 and a pharmaceutically acceptable carrier.

55. A method for treating a T cell, B ce!f, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the selected T- MSCs of Claim 43 in an amouni sufficient to ameliorate or a!ieviate at least one symptom of the diseases or reverse the diseases.

56. A method of preventing a T celi, S cell, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the selected T-MSCs of Claim 43 in an amount sufficient to prevent the diseases from developing or to minimize the extent of the disease or slow their development.

57. A method for treating a T celi, B cell, inflammatory and/or innate immunity related diseases comprising administering io a subject in need thereof th seiected T- MSCs of Claim 44 in an amount sufficient to ameliorate or alleviate at least one symptom of the diseases or reverse the diseases.

58. A method of preventing a T ceil, 8 cell, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the seiected T-MSCs of Claim 44 in an amount sufficient to prevent the diseases from developing or to minimiz the extent of the diseases or slow their development.

59. A method for treating a T celi, B celi, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the seiected T- MSCs of Claim 45 in an amount sufficient to ameliorate or a!ieviate at least one symptom of the diseases or reverse the diseases.

60. A method of preventing a T ce!S, B cell, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the selected T-MSCs of Claim 45 in an amount sufficient to prevent the diseases from developing or to minimize the extent of the diseases or siovv their development.

61 . A method for treating a T cell B celi, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof th seiected T- MSCs of Claim 46 in an amount sufficient to ameliorate or a!ieviate at least one symptom of the diseases or reverse the diseases.

62. A method of preventing a T cell, S ceil, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the selected T-MSCs of Claim 46 in an amount sufficient to prevent the diseases from developing or to minimize the extent of the diseases or slow their development.

63. A method for treating a T cell, B ceii, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the pharmaceutical preparation of Claim 51 in an amount sufficient to ameliorate or alleviate at least one symptom of the diseases or reverse the diseases.

64. A method of preventing a T ceii, B cell, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the pharmaceuticai composition of Claim 51 in an amount sufficient to prevent the diseases from developing or to minimize the extent of the diseases or slow their development.

65. A method for treating a T ceil, S cell inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the pharmaceutical preparation of Claim 52 in an amount sufficient to ameiioraie or alleviate at least one symptom of the diseases or reverse th diseases.

66. A method of preventing a T cell, B cell, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the pharmaceuticai composition of Claim 52 in an amount sufficient to prevent the diseases from developing or to minimize the extent of the diseases or slow their development.

67. A method for treating a T celi, 8 cell, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the pharmaceutical preparation of Claim 53 in an amount sufficient to ameliorate or alleviate at least one symptom of the diseases or reverse the diseases.

68. A method of preventing a T ceil, B ceil, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the pharmaceuticai composition of Claim 53 in an amount sufficient to prevent the diseases from developing or to minimize the extent of the diseases or slow their development.

69. A method for treating a T cell, B celi, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the pharmaceutica! preparation of Claim 54 in an amount sufficient to ameliorate or alleviate at least one symptom of the diseases or reverse the diseases.

70. A method of preventing a T celi, B cell, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof the pharmaceutical composition of Claim 54 in an amount sufficient to prevent the diseases from developing or to minimize the extent of the diseases or slow their development,

71. A method for treating multiple sclerosis comprising administering to a subject in need thereof the seiected T-MSCs of Claim 43 in an amount sufficient to ameliorate or alleviate at Seast one symptom of the multiple scierosis or reverse the multiple scierosis.

72. A method of preventing multiple sclerosis comprising administering to a subject in need thereof the seiected T-MSCs of Claim 43 in an amount sufficient to prevent the multiple scierosis from developing or to minimize the extent of the multiple sclerosis or slow its development.

73. A method for treating multiple sclerosis comprising administering to a subject in need thereof the seiected T-MSCs of Claim 44 in an amount sufficient to ameliorate or alleviate at least one symptom of the multiple sclerosis or reverse the multiple sclerosis.

74. A method of preventing multiple scierosis comprising administering to a subject in need thereof the seiected T-MSCs of Claim 44 in an amount sufficient to prevent the multiple scierosis from developing or to minimize the extent of the multiple sclerosis or slow its development.

75. A method for treating multiple sclerosis comprising administering to a subject in need thereof the seiected T-MSCs of Claim 45 in an amount sufficient to ameliorate or alleviate at Seast one symptom of the multiple scierosis or reverse the multiple scierosis.

76. A method of preventing multiple scierosis comprising administering to a subject in need thereof the seiected T~MSCs of Claim 45 in an amount sufficient to prevent the multiple scierosis from developing or to minimize the extent of the multiple scierosis or siow its development.

77. A method for treating multiple sclerosis comprising administering to a subject in need thereof the seiected T-MSCs of Claim 46 in an amount sufficient to ameliorate or alleviate at least one symptom of the multiple sclerosis or reverse the multiple scierosis.

78. A method of preventing multiple scierosis comprising administering to a subject in need thereof the selected T-MSCs of Claim 46 in an amount sufficient to prevent the multiple sclerosis or to minimize the extent of the multiple scierosis or slow its development.

79. A method for treating multiple scierosis comprising administering to a subject in need thereof the pharmaceutical preparation of Ciaim 51 in an amount sufficient to ameliorate or alleviate at !east one symptom of the multiple sclerosis or reverse the multiple sclerosis.

80. A method of preventing multiple sclerosis administering to a subject in need thereof the pharmaceutical composition of Claim 51 in an amount sufficient fo prevent the multiple sclerosis from developing or to minimize the extent of the multiple scierosis or slow its development.

81 . A method for treating multiple scierosis comprising administering to a subject in need thereof the pharmaceutica! preparation of Claim 52 in an amount sufficient to ameliorate or alleviate at ieast one symptom of the multiple sclerosis or reverse the multiple scierosis.

82. A method of preventing multiple sclerosis comprising administering to a subject in need thereof the pharmaceutical composition of Claim 52 in an amount sufficient to prevent the multiple sclerosis from developing or to minimize the extent of the multiple scierosis or slow its development.

83. A method for treating multiple sclerosis comprising administering to a subject in need thereof the pharmaceutica! preparation of Ciaim 53 in an amount sufficient to ameliorate or alleviate at least one symptom of the multiple scierosis or reverse the multiple sclerosis,

S4. A method of preventing multiple sclerosis comprising administering to a subject in need thereof the pharmaceutical composition of Claim 53 in an amount sufficient to prevent the multiple scierosis from developing or to minimize the extent of the multiple scierosis or slow its development

85. A method for treating multiple sclerosis comprising administering to a subject in need thereof the pharmaceutica! preparation of Claim 54 in an amount sufficient to ameliorate or aileviate at ieast one symptom of the multiple scierosis or reverse the multiple sclerosis.

86. A method of preventing multiple sclerosis comprising administering to a subject in need thereof the pharmaceutica! composition of Claim 54 in an amount sufficient to prevent the multiple sclerosis from developing or to minimize the extent of the multiple scierosis or siow its development.

87. A method of delivering an agent through the blood-brain barrier and/or the blood-spinai cord barrier, the method comprising the steps of: attaching an agent to a T- SC to form a T-MSC-agent complex; and administering the T-MSC-ageni complex to a subject in need thereof, wherein the T-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.

88. A kit comprising the selected T-MSCs of Claim 43 and a carrier.

89. The kit of C!aim 89 further comprising a thawing reagent, immunosuppressive enhancer, and anti-hisiamine.

90. The method of Claim 41, wherein the TMSCs are irradiated with gamma- if radiation.

91. The method of Claim 41 , wherein the TMSCs are irradiated with Cesium- 137 gamma irradiation or photon radiation using X-ray.

92. A method of treating or preventing cancer or tumors comprising administering to a subject in need thereof an effective amount of the pharmaceutica! composition of Claim 51.

93. The method of Claim 92, further comprising administration of a second therapeutic agent.

94. The method of Claim 92, wherein the subject was previously treated wit a therapeutic agent.

95. The method of Claim 92, wherein the se!ected T- SCs are modified by genetic modification, epigenetie regulation, sma!l molecule, nutraceutical, natural compound, or antibody treatment.

96. Modified trophoblast-derived mesenchymal stem cells (T-MSCs) produced by the method of Claim 36.

97. A ceil culture comprising the modified T-MSCs of Claim 96.

9S. A pharmaceutical preparation comprising the modified T-MSCs of Claim 96 and a pharmaceutically acceptable carrier,

99. A method for preventing or treating a T cell, 8 cell, inflammatory and/or innate immunity related diseases comprising administering to a subject in need thereof an effective amount of the modified T-MSCs produced fay the method of Claim 36.

100. A method for preventing or treating multiple sclerosis comprising administering to a subject in need thereof an effective amount of the modified T-MSCs produced by the method of Claim 36.

101. A kit comprising the modified T-MSCs of Claim 96 and a carrier.

102. The kit of Claim 101 further comprising a thawing reagent, immunosuppressive enhancer, and anti-hisfamine.

103. The modified T-MSCs of Claim 98, wherein the modified T-MSCs are further irradiated with gamma-irradiation.

104. The modified T-MSCs of Ciaim 103, wherein the irradiation is Cesium-137 gamma irradiation or photon radiation using X-ray.

105. The method of Ciaim 36, wherein the T-MSCs are modified through sh NA, mi A, knock-out, knock-in, morpho!ino, decoy RNA, D A methylation regulation, histone methyiation regulation, translation inhibition and/or antibody blocking, or a combination thereof.

108. The modified T-MSCs produced by the method of Claim 36, wherein the characteristics of the modified T-MSCs further comprise the characteristics of expressing CD73 and of iow expression or non-expression of IL-6

107. The modified T-MSC produced by the method of C!aim 36, wherein the characteristics of the modified T-MSCs further comprise the characteristics of: expressing at Ieast one ceil marker from CD90, CD105, CD13, CD29, CD54, CD146, CD 166 and CD44; iow expression or non-expression of at ieast one cell marker chosen from CD34, CD31 , and CD45; and low expression or non-expression of at least one marker from

MMP, RAGE, SFNyRI , IFNyR2, 1L-12, TNFa and VCAM1.

108. A conditioned medium, concentrate of the conditioned medium, eel! iysate or derivatives thereof, produced by the method of Ciairn 31.

109. A conditioned medium, concentrate of the conditioned medium, ceii Iysate or derivatives thereof, produced by the method of Ciairn 36.

110. The method of Claim 36, wherein the MSG is BM-MSC, T-MSC, hES- SC or adult tissue derived MSC.

11 1. A method of co-cuituring the T-MSCs of Claim 43 with bone marrow hematopoietic stem eels and/or umbilical-cord hematopoietic stem ceiis.

112. The method of Claim 1 11 , wherein the T-MSCs express Stro-3.

113. The method of Ciairn 1 11 , wherein the T-MSCs express Stro-1 ,

114. The method of Claim 1 11 , wherein the T-MSCs express Nestin,

115. The method of Ciairn , wherein the T-MSCs are mesenchymal stroma! cells.

116. A co-cuiture of the T-MSCs of Claim 43 and bone marrow hematopoietic stem cells.

117. A co-culture of the T-MSCs of Ciairn 43 and umbilical-cord hematopoietic stem cells.

118. A co-culture of hES-MSC of Claim 43 and peripheral blood hematopoietic stem celis.

1 19. The method of Claim 31, wherein the characteristics of the selected T- MSCs are determined by flow cytometry, muitipiex microarray, RT-PCT, northern blot or western blot.

120. The method of Claim 36, wherein the characteristics of the T-MSCs are determined by flow cytometry, multiplex microarray, RT-PCT, northern biot and western blot.

121 A method for using trophobiast-derived mesenchymal stem eels T- MSCs) for tissue regeneration comprising administering to a subject in need thereof the selected T-MSCs of Claims 43-46 and Claims 51-54 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 muscie regeneration, epitheiia tissue regeneration, and/or ligament regeneration.

122. A method for using trophobiast-derived mesenchymal stem eels (T- MSCs) for tissue repair comprising administering to a subject in need thereof the selected T-MSCs of Claim 43-46 and Claims 51 -54 in an amount sufficient to promote healings including, but not limited to, joint healing, tendon healing, connective tissue healing, neural lineage cells healing, fat tissue healing, bone healing, skin healing, other wound healing, muscie healing, cartilage healing, smooth muscle healing, cardiac muscle healing, epitheiia tissue healing, and/or ligament healing.

123. A method for using trophobiast-derived mesenchymal stem cells (T- SCs) for acute tissue injury treatment comprising administering to a subject in need thereof the selected T-MSCs of Claims 43-46 and Claims 51-54 in an amount sufficient to treat multiple acute injury conditions including, but not limited to, spinal cord injury, acute heart infarction, acute radiation syndrome, acute burn, acute fracture, acute soft tissue injury, and/or acut organ injuries,

124. A method of producing cell lineages differentiated from trophobiast- derived mesenchymal stem eels (T-MSCs), the cell lineages hereinafter referred to as T- MSC-DL, the method comprising:

(a) plating the T-MSCs onto gelatin, vitronectin, laminin, ftbronectin, atrigel or collagen-coated plates plated at a concentration of 1 * 10³ cells/cm² to 1 ^χ iQ ceils/cn ; and

(b) culturing the T-MSCs for a suitable time period in a serum free medium or serum-containing medium containing bovine serum FBS or human AB serum ABHS to produce T-MSC-DL.

125. A method of producing cell lineages differentiated from trophobiast- derived mesenchymal stem cells (T-MSCs), the cell lineages hereinafter referred to as T- MSC-DL, the method comprising culturing the T-fv!SCs for a suitable time period in a medium comprising 1-50 ng/ml, preferably 10 ng/ml; Fibroblast Growth Factor (FGF)-2; 1-50 ng/ml, preferably 10 ng/ml, Epidermal Growth Factor (EGF); and 0.5-5 ng/ml, preferably 1 ng/ml, Platelet-Derived Growth Factor (PDGF) to produce T-MSC-DL,

128. A method of producing a dopaminergic neuronal phenotype celi lineage differentiated from trophoblast-derived mesenchymal stem ceiis (T-MSCs), the method comprising the steps of;

(a) cuitunng the T-MSCs for a suitable time period in a medium comprising 1-50 ng/ml, preferably 10 ng/mi, Fibroblast Growth Factor {FGF)~2, and 1-50 ng/mi, preferably 10 ng/mi, Epidermal Growth Factor (EGF) to prime T-MSCs towards a neural fate;

(b) cuitunng the T-MSCs from step (a) in a medium comprising 10- 200 ng/mi, preferably 100 ng/mi, Sonic Hedgehog; (SHH); 1-50 ng/ml, preferabiy 10 ng/mi, FGF-8 (human); and 50-500 μΜ, preferably 200 μΜ, AAP to initiate midbrain specification; and

(c) cuituring the T-MSCs from step (b) in a medium comprising 5-500

ng/ml, preferably 50 ng/mi, Giial-Derived Neurotrophic Factor (GDNF), and 50-500 μΜ, preferably 200 μΜ, AAP to induce differentiation and maturation of the T-MSCs into a dopaminergic neuronal phenotype celi iineage,

127. A method of producing an osteogenic phenotype celi lineage differentiated from trophoblast-derived mesenchymal stem ceiis (T-MSCs), the method comprising cuituring the T-MSCs for a suitable time period in a medium comprising low glucose DMEM plus 10% PCS; 1-150 μΜ, preferably 80 μΜ, ascorbic acid 2- phosphate; 0,5-5 μ , preferably 1 μΜ, dexamethasone; and 1-100 mM, preferably 20 mM, beta- glycerophosphate to induce osteogenic differentiation of T-MSCs.

128. A method of producing an adipogenic phenotype cell Iineage differentiated from trophoblast-derived mesenchymal stem ceils (T-MSCs), the method comprising cuituring the T-MSC for a suitable time period in a medium comprising low glucose DMEM pius 20% FCS; 1-10 pg/mS, preferably 5 pg/mi, insulin; 0.5-10 μΜ, preferabiy 2 μΜ, dexamethasone; 0.1-1 mM, preferably 0,5 mM, isobutyimethyixanthine; and 1-100 μΜ, preferabiy 60 μΜ, indomethaein to induce adipogenic differentiation of T- MSCs.

129. A method of producing a chondrogenic phenotype eel! Iineage differentiated from trophoblast-derived mesenchymal stem ceils (T-MSCs), the method comprising cuituring the T-MSCs for a suitable time period in a peiief in a medium comprising high glucose DMEM plus 0.5-10 mM, preferabiy 1 mM, Sodium Pyruvate; 0.5- 1 mM, preferabiy 0.1 mM, ascorbic acid 2- phosphate; 0.05-1 μΜ, preferably 0.1 μΜ, dexamethasone; 0.2-2%, preferably 1%, ITS; and 1 -50 ng/ml, preferably 10 ng/mi, TGF- β3 to induce chondrogenic differentiation of T-MSCs,

130. A method of producing a mycogenic phenotype ceil lineage differentiated from trophobSasi-derived mesenchymal stem cells (T-MSCs), the method comprising the steps of:

(a) cuituring the T-MSCs for a suitable time period in a medium comprising low glucose DMEM plus 10% FBS; 1 -20 μΜ, preferably 10 μΜ, 5-azacytidine; and 1 -50 ng/ml, preferably 10 ng/ml, basic FGF for 24 hours;

(b) replacing the medium of step (a) with medium comprising D EM

plus 10% FBS and 1 -50 ng/ml, preferably 10 ng/ml, basic FGF to induce mycogenic differentiation of T-MSCs.

131. A method of producing a fibroblast phenotype eel! lineage differentiated from trophobiast-derived mesenchymal stem ceiis (T-MSCs), th method comprising cuituring the T-MSCs for a suitable time period in a medium comprising DMEM plus 10% FBS; 50-200 ng/ml, preferably 100 ng/ml, of recombinant human Connective Tissue Growth Factor (CTGF); and 1-100 pg/ i, preferably 50 pg/mi, ascorbic acid to induce fibroblast differentiation of T-MSCs.

132. A method of producing cell lineages differentiated from trophobiast- derived mesenchymal stem cells (T-MSCs), the cell lineages hereinafter referred to as T- MSC-DL, the method comprising the steps of;

(a) cuituring the T-MSCs for a suitable time period in a medium comprising Neurobasa! medium plus 0.25 x 8-27 supplement; 10- 200 ng/mi, preferably 100 ng/mi, Sonic Hedgehog (SHH); 1 -50 ng/ml, preferably 10 ng/mi, FGF-8 (mouse); and 1-200 ng/mi, preferably 50 ng/ml, FGF-2; and

(b) harvesting the T-MSC-DL after cuituring the T-MSCs of step (a)

for 6 to 12 days.

133. A method of producing ceil lineages differentiated from trophobiast- derived mesenchymal stem cells (T-MSCs), the cell lineages hereinafter referred to as T- MSC-Dl, the method comprising the steps of:

(a) cuituring the T-MSCs in a medium comprising serum-free medium

(DMEM) plus 2 mM glutamine; 1 -20 U/ml, preferably 12.5 U/mi, nystatin; N2 supplement; 2-50 ng/ml, preferably 20 ng/mi, fibroblast growth factor-2 (FGF-2); and 1-50 ng/ml, preferably 10 ng/ml, EGF for 48 to 72 hours; and

(b) cuituring the T-MSCs of ste (a) in a medium comprising a

Neurobasa! medium plus B27 supplement; 0.1 -10 mM, preferably 1 mM, dibutyry! cyclic AMP (dbcAMP); 3-isobutyi-1- methyixanthtne (!BMX); 10-500 μΜ, preferably 200 μΜ, ascorbic acid; 1 -100 ng/mi, preferably 50 ng/mi, BDNF; 1 -50 ng/ml, preferabiyi O ng/mi, giiai-derived neurotrophic factor (GDNF); 0.2- 10 ng/mi, preferabiy 2 ng/mi, transforming growth factor~/33 (TGF- 5 jS3); and 0.05-5 μΜ, preferably 0.1 μΜ₍ all-iransretinoic acid (RA) to produce T- SC-DL