NZ709113B2

NZ709113B2 - Anoikis resistant placental stem cells and uses thereof

Info

Publication number: NZ709113B2
Application number: NZ709113A
Authority: NZ
Inventors: Stewart Abbot; Vanessa Voskinarianberse; Xiaokui Zhang; Berse Vanessa Voskinarian
Original assignee: Celularity Inc
Priority date: 2012-12-14
Filing date: 2013-12-13
Publication date: 2021-03-02

Abstract

Anoikis resistant placental stem cells ( arPSCs) with increased survival in low-attachment environments, and thus can advantageously be used, e.g., in therapies based on their ability to persist for longer durations of time in an unattached state. A method of modifying placental stem cells to make them anoikis resistant, comprising contacting the placental stem cells with an effective amount of modulatory RNA molecules, such that one or more genes associated with anoikis of the placental stem cells is inhibited. Further discloses are genes that are associated with anoikis for modulation. In a specific embodiment, the modulated genes are any one of FHDC1, GNAI2, KNDC1, LPAR4, MAP3K5, SLC2A3, or STAU2. hem anoikis resistant, comprising contacting the placental stem cells with an effective amount of modulatory RNA molecules, such that one or more genes associated with anoikis of the placental stem cells is inhibited. Further discloses are genes that are associated with anoikis for modulation. In a specific embodiment, the modulated genes are any one of FHDC1, GNAI2, KNDC1, LPAR4, MAP3K5, SLC2A3, or STAU2.

Description

/074892 ANOIKIS RESISTANT PLACENTAL STEM CELLS AND USES THEREOF This ation claims priority to US. Provisional Patent Application No. 61/737,498, ﬁled December 14, 2012, the disclosure of which is orated herein by reference in its entirety. 1. FIELD Provided herein are s-resistant placental cells and compositions thereof as well as methods of using such cells and compositions. 2. BACKGROUND Because mammalian placentas are plentiﬁll and are normally ded as medical waste, they represent a unique source of medically-useful cells, e. g., placental stem cells.

Placental stem cells, typically adhere (attach) to culture surfaces, such as tissue culture plates and extracellular . Anoikis is a form of programmed cell death (apoptosis) that occurs in attachment-dependent cells when they are cultured/present in low attachment environments.

There exists a need for populations of placental stem cells that are resistant to anoikis, and thus e for longer periods of time in a non-adherent state. Provided herein are such improved placental stem cells, populations of such tal stem cells, and methods of using the same. 3. SUMMARY In one aspect, provided herein is a method of modifying placental stem cells to make them anoikis resistant. The anoikis resistant placental stem cells (arPSCs) provided herein demonstrate increased survival in low-attachment environments, and thus can advantageously be used, e.g., in therapies that utilize administration of placental stem cells (e.g., ic administration of placental stem cells) based on their ability to persist for longer durations of time in an unattached state, e.g., as compared to unmodified tal stem cells (e.g., placental stem cells that have not been modified to be anoikis resistant). In certain embodiments, placental stem cells are s resistant if they are capable of surviving in conditions in which placental stem cells would normally undergo anoikis. In n embodiments, placental stem cells are anoikis resistant if they are capable of surviving for a longer duration of time relative to unmodiﬁed placental stem cells in conditions in which placental stem cells would normally undergo anoikis.

In one embodiment, provided herein is a method of ing placental stem cells to make them anoikis resistant, comprising contacting the placental stem cells with an effective amount of oligomeric or polymeric molecules, such that one or more genes associated with anoikis of the placental stem cells is inhibited (e.g., downregulated as ed to placental stem cells that have not been modified, e.g., that have not been contacted with said molecules).

Such modified placental stem cells described herein are referred to herein as “anoikis resistant placental stem cells” (“arPSCs”). In certain embodiments, said oligomeric or polymeric molecules are modulatory RNA molecules. In specific embodiments, the modulatory RNA molecules are small ering RNAs s), microRNA inhibitors (miR inhibitors), miR , antisense RNAs, small hairpin RNAs (shRNAs), microRNA-adapted shRNA (shRNAmirs), or any combination thereof.

In certain embodiments, the tory RNA molecules used in the methods described herein for generating arPSCs target one or more placental stem cell genes (“anoikis- associated genes”) identified herein as being associated with anoikis in the placental stem cells.

In a specific embodiment, said one or more anoikis-associated genes targeted in the methods described herein to produce arPSCs comprise one or more of the genes listed in Table 1, below: Table 1: Human Placental Stem Cell Anoikis Associated Genes Gene ID Gene Symbol Gene Description (NCBI) 57463 AMIGOl adhesion molecule with Ig-like domain 1 57569 ARHGAP20 Rho GTPase activating protein 20 952 CD38 CD38 molecule 23155 CLCCl de l CLIC-like l 1270, CNTF, ZFP9 l - ciliary rophic factor| ZFP9 l -CNTF 3 86607 CNTF readthrough ript l351 COX8A cytochrome c oxidase subunit 8A (ubiquitous) 9704 DHX34 DEAH (Asp-Glu-Ala—His) box polypeptide 34 1023 FAM 1 75A, mitochondrial ribosomal protein S l 8C| family 84 142 MRPS 1 8C with sequence similarity 175, member A Gene ID Gene Symbol Gene Description (NCBI) 284257 FAM44C family With sequence similarity 44, member C 8789 FBP2 fructose-1,6-bisphosphatase 2 2313 FLI1 Friend leukemia Virus integration 1 166752 FREM3 FRASl related extracellular matrix 3 24138 IFIT5 interferon-induced protein With tetratricopeptide repeats 5 399851 LOC399851 hypothetical gene supported by AY129010 400713 LOC400713 zinc ﬁnger-like 651610 LOC651610 serine-protein kinase ATM-like 51227 PIGP phosphatidylinositol glycan anchor biosynthesis, class P 79628 SH3TC2 SH3 domain and tetratricopeptide repeats 2 6515 SLC2A3 solute carrier family 2 (facilitated glucose transporter), member 3 27067 STAU2 n, RNA binding n, homolog 2 (Drosophila) 8577 TMEFF1 transmembrane protein With EGF-like and two follistatin-like domains 1 221468 TMEM217 transmembrane protein 217 84283 TMEM79 transmembrane n 79 83878 USHBP1 Usher syndrome 1C binding protein 1 83464 APH1B or pharynx defective 1 g B (C. elegans) 491 ATP2B2 ATPase, Ca++ transporting, plasma membrane 2 196541 C13orf39 chromosome 13 open reading frame 39 84103 C4orf17 chromosome 4 open reading frame 17 201725 6 chromosome 4 open reading frame 46 51428 DDX41 DEAD (Asp-Glu-Ala-Asp) box polypeptide Gene ID Gene Symbol Gene Description (NCBI) 84237 DKFZp547J222 hypothetical LOC8423 7 2260 FGFR1 ﬁbroblast growth factor receptor 1 85462 FHDC1 FH2 domain containing 1 2771 GNAI2 guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 2814 GP5 rotein V (platelet) 3557 IL1RN interleukin 1 receptor antagonist 347240 KIF24 n family member 24 85442 KNDC1 kinase non-catalytic C-lobe domain (KIND) containing 1 10013259 LOC100132598 similar to hCG2001 192 151760 LOC151760 hypothetical LOC151760 152024 LOC152024 hypothetical protein LOC152024 339833 LOC339833 hypothetical protein LOC339833 2846 LPAR4 lysophosphatidic acid receptor 4 55341 LSG1 large t GTPase 1 g (S. cereVisiae) 4217 MAP3K5 n-activated protein kinase kinase kinase 5165 PDK3 pyruvate dehydrogenase kinase, isozyme 3 57161 PELI2 pellino homolog 2 (Drosophila) 7844 RNF103 ring ﬁnger protein 103 169166 SNX31 sorting nexin 31 25828 TXN2 thi0red0xin 2 343702 XKR7 XK, Kell blood group complex subunit-related Gene ID Gene Symbol Gene Description (NCBI) related family, member 7 [006a] In particular aspects of the present invention said anoikis associated gene is FHDC1 (NCBI GENE ID NO:85462), GNAI2 (NCBI GENE ID NO:2771), KNDC1 (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID 7), SLC2A3 (NCBI GENE ID NO:6515), or STAU2 (NCBI GENE ID NO:27067). [006b] Thus in one aspect the t invention provides an isolated placental stem cell, wherein said placental stem cell is resistant to anoikis, n said placental stem cell expresses at least one anoikis associated gene at a decreased level as compared to the expression of the same anoikis associated gene in an unmodified placental stem cell, and wherein said anoikis associated gene is FHDC1 (NCBI GENE ID NO:85462), GNAI2 (NCBI GENE ID NO:2771), KNDC1 (NCBI GENE ID 42), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), or STAU2 (NCBI GENE ID NO:27067). In certain embodiments the s resistant placental stem cell is a CD10+, CD34-, CD105+, CD200+ placental stem cell. [006c] In another aspect the invention particularly es an isolated population of cells comprising anoikis resistant placental stem cells wherein said anoikis resistant placental stem cells express at least one anoikis associated gene at a decreased level as compared to the expression of the same anoikis associated gene in an unmodified placental stem cell, and wherein said anoikis associated gene is FHDC1 (NCBI GENE ID 62), GNAI2 (NCBI GENE ID NO:2771), KNDC1 (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), or STAU2 (NCBI GENE ID NO:27067). In certain embodiments the anoikis resistant placental stem cell is a CD10+, CD34-, CD105+, CD200+ placental stem cell. In certain embodiments at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, and least 85%, at least 90%, at least 95%, or at least 99% of the cells in said population of cells are s resistant placental stem cells. [006d] In a still further aspect the invention provides method of ing anoikis resistant placental stem cells comprising contacting the placental stem cells with an ive amount of modulatory RNA molecules, such that said placental stem cells, after having been ted with said modulatory RNA molecules s at least one anoikis associated gene at a decreased level as compared to the expression of the same anoikis associated gene in an equivalent amount (followed by page 5A) of placental stem cells not contacted with said modulatory RNA molecules, wherein said anoikis associated gene is FHDC1 (NCBI GENE ID NO:85462), GNAI2 (NCBI GENE ID 1), KNDC1 (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), or STAU2 (NCBI GENE ID NO:27067), and provided said contacting is not within a human. In certain embodiments the anoikis resistant placental stem cell is a CD10+, CD34-, CD105+, CD200+ tal stem cell.

In one embodiment, the modulatory RNA molecules used in the s described herein for generating arPSCs are small interfering RNAs (siRNAs). In a specific embodiment, said siRNAs target one or more of the anoikis-associated genes listed in Table 1, above. In another specific embodiment, said siRNAs are double-stranded, wherein one strand of said siRNAs have a sequence at least about 70%, 80%, 90%, 95%, 98% or 100% complementary to the sequence of one of the genes identified in Table 1, above (as identified based on the Gene ID of the gene provided in the table).

In another specific embodiment, the siRNAs used in the methods described herein for ting arPSCs target the placental stem cell anoikis associated gene FHDC1 (NCBI GENE ID NO:85462). In another specific embodiment, the siRNAs used in the s described herein for generating arPSCs target the placental stem cell anoikis associated gene GNAI2 (NCBI GENE ID NO:2771). In another specific embodiment, the siRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene KNDC1 (NCBI GENE ID NO:85442). In another specific ment, the siRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis ated gene LPAR4 (NCBI GENE ID 6). In another specific embodiment, the siRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene MAP3K5 (NCBI GENE ID NO:4217). In another specific embodiment, the siRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene SLC2A3 (NCBI GENE ID NO:6515). In another specific embodiment, the siRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene STAU2 (NCBI GENE ID NO:27067).

In another specific embodiment, the siRNAs used in the s described herein for generating arPSCs target one, two, three, or more of the following tal stem cell s-associated genes: FHDC1 (NCBI GENE ID NO:85462), GNAI2 (NCBI GENE ID NO:2771), KNDC1 (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID 7), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (followed by page 6) (NCBI GENE ID NO:27067). In another speciﬁc ment, the siRNAs used in the methods described herein for ting arPSCs target one, two, three, or more of the following placental stem cell anoikis-associated genes: FHDC1 (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID NO:2771), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067), and target at least one additional anoikis ated gene recited in Table 1.

In another embodiment, the tory RNA molecules used in the methods described herein for generating arPSCs are small hairpin RNAs s). In a speciﬁc embodiment, said shRNAs target one or more of the anoikis-associated genes listed in Table 1, above. In another speciﬁc embodiment, said shRNAs have a sequence at least about 70%, 80%, 90%, 95%, 98% or 100% complementary to the sequence of one of the genes identiﬁed in Table 1, above (as identiﬁed based on the Gene ID of the gene provided in the table).

In another speciﬁc embodiment, the shRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene FHDC1 (NCBI GENE ID NO:85462). In another speciﬁc embodiment, the shRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene GNA12 (NCBI GENE ID NO:2771). In r speciﬁc embodiment, the shRNAs used in the methods described herein for ting arPSCs target the placental stem cell anoikis associated gene KNDC1 (NCBI GENE ID NO:85442). In another speciﬁc embodiment, the shRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene LPAR4 (NCBI GENE ID NO:2846). In another speciﬁc embodiment, the shRNAs used in the s described herein for generating arPSCs target the placental stem cell s associated gene MAP3K5 (NCBI GENE ID NO:4217). In another speciﬁc embodiment, the shRNAs used in the methods described herein for generating arPSCs target the placental stem cell s associated gene SLC2A3 (NCBI GENE ID NO:6515). In r speciﬁc embodiment, the shRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene STAU2 (NCBI GENE ID NO:27067).

In another speciﬁc embodiment, the shRNAs used in the methods described herein for generating arPSCs target one, two, three, or more of the ing placental stem cell anoikis-associated genes: FHDC1 (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID NO:2771), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAUZ (NCBI GENE ID NO:27067). In another speciﬁc embodiment, the shRNAs used in the methods described herein for generating arPSCs target one, two, three, or more of the following placental stem cell anoikis-associated genes: FHDC1 (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID NO:2771), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067), and target at least one additional anoikis associated gene recited in Table 1.

In another ment, the modulatory RNA molecules used in the s described herein for generating arPSCs are antisense RNAs. In a speciﬁc embodiment, said nse RNAs target one or more of the anoikis-associated genes listed in Table 1, above. In another c ment, said antisense RNAs have a sequence at least about 70%, 80%, 90%, 95%, 98% or 100% complementary to the sequence of one of the genes identiﬁed in Table 1, above (as identiﬁed based on the Gene ID of the gene provided in the table).

In another speciﬁc embodiment, the antisense RNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene FHDC1 (NCBI GENE ID NO:85462). In another speciﬁc embodiment, the antisense RNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene GNAIZ (NCBI GENE ID 1). In another speciﬁc embodiment, the antisense RNAs used in the methods bed herein for generating arPSCs target the tal stem cell anoikis associated gene KNDCl (NCBI GENE ID NO:85442). In another speciﬁc embodiment, the antisense RNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene LPAR4 (NCBI GENE ID NO:2846). In another speciﬁc embodiment, the antisense RNAs used in the methods bed herein for generating arPSCs target the placental stem cell anoikis associated gene MAP3K5 (NCBI GENE ID NO:4217), In another speciﬁc embodiment, the antisense RNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene SLC2A3 (NCBI GENE ID NO:6515). In another speciﬁc embodiment, the antisense RNAs used in the methods bed herein for generating arPSCs target the tal stem cell anoikis ated gene STAUZ (NCBI GENE ID NO:27067).

In another speciﬁc embodiment, the antisense RNAs used in the methods described herein for generating arPSCs target one, two, three, or more of the following placental stem cell anoikis-associated genes: FHDC1 (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID NO:2771), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID 67). In another speciﬁc embodiment, the antisense RNAs used in the methods described herein for generating arPSCs target one, two, three, or more of the ing placental stem cell anoikis-associated genes: FHDCl (NCBI GENE ID 62), GNA12 (NCBI GENE ID NO:2771), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067), and target at least one additional anoikis associated gene recited in Table 1.

In another embodiment, the modulatory RNA molecules used in the methods described herein for generating arPSCs target one or more microRNAs (miRNAs) in placental cells that act to modulate the production of one or more anoikis-associated genes. In one embodiment, said modulatory RNA molecules are miR inhibitors. In another embodiment, said modulatory RNA les are miR mimics. In a speciﬁc embodiment, the miRNA ed is an miRNA that modulates one or more of the anoikis-associated genes listed in Table 1, above.

In certain embodiments, said miR inhibitors or said miR mimics have a sequence at least about 70%, 80%, 90%, 95%, 98% or 100% complementary to the sequence an miRNA that modulates the production of one of the genes identiﬁed in Table 1.

In another c embodiment, the miR inhibitors or miR mimics used in the methods described herein for generating arPSCs target a miRNA that modulates the tion of the placental stem cell anoikis associated gene FHDCl (NCBI GENE ID NO:85462). In another speciﬁc embodiment, the miR tors or miR mimics used in the methods described herein for generating arPSCs target a miRNA that modulates the production of the placental stem cell anoikis associated gene GNAI2 (NCBI GENE ID NO:2771). In another speciﬁc embodiment, the miR inhibitors or miR mimics used in the methods described herein for ting arPSCs target a miRNA that modulates the production of the placental stem cell anoikis associated gene KNDCl (NCBI GENE ID 42). In another speciﬁc embodiment, the miR inhibitors or miR mimics used in the methods described herein for generating arPSCs target a miRNA that modulates the tion of the placental stem cell anoikis associated gene LPAR4 (NCBI GENE ID NO:2846). In another speciﬁc embodiment, the miR inhibitors or miR mimics used in the s described herein for generating arPSCs target a miRNA that modulates the production of the placental stem cell anoikis associated gene MAP3K5 (NCBI GENE ID NO:4217). In another c embodiment, the miR inhibitors or miR mimics used in the methods described herein for generating arPSCs target a miRNA that modulates the production of the tal stem cell anoikis associated gene SLC2A3 (NCBI GENE ID ). In another speciﬁc embodiment, the miR inhibitors or miR mimics used in the methods described herein for generating arPSCs target a miRNA that modulates the production of the tal stem cell anoikis associated gene STAU2 (NCBI GENE ID NO:27067).

In another speciﬁc embodiment, the miR inhibitors or miR mimics used in the methods described herein for generating arPSCs target one, two, three, or more miRNAs, wherein said miRNAs modulate the production of one, two, three, or more of the following placental stem cell anoikis-associated genes: FHDCl (NCBI GENE ID NO:85462), GNAIZ (NCBI GENE ID NO:277l), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067). In another speciﬁc embodiment, the miR inhibitors or miR mimics used in the methods described herein for generating arPSCs target one, two, three, or more miRNAs, wherein said miRNAs te the production of one, two, three, or more of the following placental stem cell anoikis-associated genes: FHDCl (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID NO:277l), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067), and target at least one miRNA that modulates the production of at least one additional anoikis associated gene recited in Table l.

In another , ed herein are isolated anoikis ant placental stem cells (arPSCs), and compositions thereof, produced ing to the methods described herein, e.g., placental stem cells that have been modiﬁed by contacting said placental stem cells with an effective amount of oligomeric or polymeric molecules (e.g., modulatory RNA molecules), to render them anoikis resistant. Such anoikis resistant placental stem cells demonstrate increased survival in low-attachment nments as compared to, e.g., unmodiﬁed placental stem cells (e. g., placental stem cells that have not been contacted with an ive amount of oligomeric or polymeric les (e.g., modulatory RNA molecules)).

In one embodiment, the isolated arPSCs provided herein express at least one anoikis associated gene at a decreased level as compared to the expression of the same s associated gene in an unmodiﬁed tal stem cell. In a c embodiment, provided herein is an isolated arPSC, or population thereof, wherein said isolated arPSC expresses at least one gene from those listed in Table l at a sed level as compared to the expression of the same anoikis associated gene in an unmodiﬁed placental stem cell. In r speciﬁc embodiment, provided herein is an isolated arPSC, or population thereof, wherein said isolated arPSC expresses at more than one gene from those listed in Table l at a decreased level as ed to the expression of the same anoikis associated gene in an ﬁed placental stem cell, e.g., the isolated arPSC expresses, two, three, four, ﬁve, six, seven, eight, nine, ten, or greater than ten genes from those listed in Table l at a decreased level as compared to the expression of the same anoikis associated gene in an unmodiﬁed placental stem cell.

In another speciﬁc embodiment, provided herein is an isolated arPSC, wherein said arPSC expresses the s associated gene FHDCl (NCBI GENE ID NO:85462) at a decreased level as compared to the expression of the anoikis associated gene FHDCl (NCBI GENE ID NO:85462) in an unmodiﬁed placental stem cell. In another speciﬁc embodiment, provided herein is an isolated arPSC, wherein said arPSC expresses the anoikis associated gene GNAIZ (NCBI GENE ID l) at a decreased level as compared to the expression of the anoikis associated gene GNA12 (NCBI GENE ID NO:277l) in an unmodiﬁed placental stem cell. In another speciﬁc embodiment, provided herein is an isolated arPSC, n said arPSC expresses the anoikis associated gene KNDCl (NCBI GENE ID NO:85442) at a decreased level as compared to the expression of the anoikis associated gene KNDCl (NCBI GENE ID 42) in an unmodiﬁed placental stem cell. In another speciﬁc embodiment, provided herein is an isolated arPSC, wherein said arPSC expresses the anoikis associated gene LPAR4 (NCBI GENE ID NO:2846) at a decreased level as compared to the expression of the anoikis associated gene LPAR4 (NCBI GENE ID NO:2846) in an unmodiﬁed tal stem cell. In another speciﬁc embodiment, provided herein is an isolated arPSC, wherein said arPSC expresses the anoikis associated gene MAP3K5 (NCBI GENE ID NO:4217) at a decreased level as compared to the expression of the anoikis associated gene MAP3K5 (NCBI GENE ID NO:4217) in an ﬁed placental stem cell. In another speciﬁc embodiment, provided herein is an isolated arPSC, wherein said arPSC expresses the anoikis associated gene SLC2A3 (NCBI GENE ID NO:6515) at a decreased level as compared to the expression of the anoikis associated gene SLC2A3 (NCBI GENE ID NO:6515) in an unmodiﬁed placental stem cell. In another speciﬁc embodiment, provided herein is an isolated arPSC, wherein said arPSC expresses the anoikis associated gene STAU2 (NCBI GENE ID NO:27067) at a decreased level as compared to the expression of the anoikis associated gene STAU2 (NCBI GENE ID NO:27067) in an ﬁed placental stem cell. Further provided herein are populations of cells comprising such arPSCs and compositions comprising such .

In another speciﬁc embodiment, provided herein is an isolated arPSC, wherein said arPSC expresses one, two, three, or more of the following placental stem cell anoikis- associated genes at a decreased level as compared to the expression of the same anoikis associated gene(s) in an unmodiﬁed placental stem cell: FHDCl (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID 1), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067). In another speciﬁc embodiment, provided herein is an ed arPSC, wherein said arPSC (i) expresses one, two, three, or more of the following placental stem cell anoikis-associated genes at a decreased level as compared to the sion of the same anoikis associated gene(s) in an unmodiﬁed placental stem cell: FHDCl (NCBI GENE ID 62), GNA12 (NCBI GENE ID NO:2771), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067); and (ii) expresses at least one additional anoikis associated gene recited in Table 1 at a decreased level as compared to the expression of the same anoikis associated gene(s) in an ﬁed placental stem cell. Further provided herein are tions of cells comprising such arPSCs and compositions comprising such .

In a speciﬁc embodiment, the arPSCs described herein are CD10+, CD347, CD105+, and CD200+. In another c embodiment, the arPSCs bed herein express CD200 and do not express HLA-G; or express CD73, CD105, and CD200; or express CD200 and OCT-4; or express CD73 and CD105 and do not express HLA-G; or express CD73 and CD105 and facilitate the ion of one or more embryoid-like bodies in a population of placental cells comprising said stem cell when said population is cultured under conditions that allow for the formation of an embryoid-like body; or express OCT-4 and facilitate the ion of one or more embryoid-like bodies in a population of placental cells comprising said stem cell when said population is cultured under conditions that allow for the formation of an embryoid- like body. In another specific embodiment, the arPSCs described herein are additionally CD90+ and CD45_. In another specific embodiment, the arPSCs described herein are additionally CD80_ and CD86_. In yet other embodiments, the arPSCs bed herein express one or more of CD44, CD90, B,C or ABC-p, and/or do not express one or more of CD45, CD1 17, CDl33, KDR, CD80, CD86, HLA-DR, SSEA3, SSEA4, or CD38. In certain embodiments, the arPSCs described herein suppress the activity of an immune cell, e.g., suppress proliferation of a T cell to a detectably greater degree than unmodiﬁed placental stem cells (e.g., placental cells that have not been contacted with an effective amount of oligomeric or polymeric molecules (e. g., modulatory RNA les)), as determinable by, e.g., a mixed leukocyte on assay, regression assay, or bead T cell assay.

In another aspect, provided herein is a method for an immune response, e.g., modulating the immune response of a subject, e.g., a human subject, or modulating an immune response in vitro, sing contacting immune cells with the arPSCs described herein, or a composition f In a specific embodiment, the arPSCs provided herein are capable of ting an immune response to the same degree as an equivalent amount of unmodified placental stem cells (e. g., placental stem cells that are not resistant to anoikis). Assays for measuring the ability of cells (e. g., placental stem cells, including arPSCs) to modulate an immune se are known in the art (see, e. g., US. Patent No. 7,682,803, the disclosure of which is herein incorporated by reference in its entirety) and described herein, e.g., mixed lymphocyte reaction, regression assay.

In another aspect, provided herein is a method for ing enesis. In a speciﬁc embodiment, provided herein is a method for promoting enesis in a subject, e. g., a human t, comprising administering to said subject the arPSCs described herein, or a composition thereof. In another specific embodiment, the arPSCs provided herein are capable of promoting angiogenesis to the same degree as an equivalent amount of unmodified placental stem cells (e. g., placental stem cells that are not resistant to anoikis). Assays for measuring the ability of cells (e.g., placental stem cells, including arPSCs) to e angiogenesis are known in the art (see, e.g., US. Patent Application Publication No. 2011/0250182, the sure of which is herein incorporated by reference in its entirety), e.g., assaying the ability of cells to promote tube formation by endothelial cells, assaying the ability of cells to promote endothelial cell ion and/or proliferation, and assaying the ability of cells to secrete factors that promote angiogenesis. 3.1 DEFINITIONS As used herein, the term “amount,” when referring to placental stem cells, e.g., anoikis resistant placental stem cells described herein, means a particular number of placental stem cells (e.g., anoikis resistant placental stem cells).

As used herein, the term “derived” means isolated from or otherwise purif1ed.

For example, tal derived adherent cells are isolated from placenta. The term “derived” encompasses cells that are cultured from cells isolated directly from a , e.g., the placenta, and cells cultured or expanded from primary isolates.

As used herein, “immunolocalization” means the detection of a compound, e.g., a cellular marker, using an immune protein, e.g., an antibody or fragment thereof in, for example, ﬂow cytometry, ﬂuorescence-activated cell sorting, magnetic cell sorting, in situ hybridization, immunohistochemistry, or the like.

As used herein, the term “SH2” refers to an antibody that binds an epitope on the marker CD105. Thus, cells that are referred to as SH2+ are CD105+.

As used herein, the terms “SH3” and SH4” refer to dies that bind epitopes present on the marker CD73. Thus, cells that are referred to as SH3+ and/or SH4+ are CD73” As used herein, a stem cell is ted” if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the other cells with which the stem cell is naturally ated are removed from the stem cell, e.g., during collection and/or culture of the stem cell. A population of “isolated” cells means a population of cells that is substantially ted from other cells of the tissue, e.g., ta, from which the population of cells is derived. In some ments, a population of, e.g., stem cells is “isolated” if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the cells with which the tion of stem cells are naturally associated are removed from the population of stem cells, e.g., during collection and/or culture of the population of stem cells.

As used herein, the term “placental stem cell” refers to a stem cell or progenitor cell that is derived from, e.g., isolated from, a mammalian placenta, regardless of the number of passages after a primary e, which s to a tissue culture substrate (e.g., tissue e plastic or a ﬁbronectin-coated tissue culture plate) in its unmodiﬁed state. The term “placental stem cell” as used herein does not, however, refer to a trophoblast, a ophoblast, embryonic germ cell, or embryonic stem cell, as those cells are understood by persons of skill in the art.

The terms “placental stem cell” and “placenta-derived stem cell” may be used interchangeably.

Unless otherwise noted herein, the term “placental” includes the umbilical cord. The placental stem cells disclosed herein are, in certain embodiments, multipotent in vitro (that is, the cells differentiate in vitro under differentiating conditions), multipotent in viva (that is, the cells differentiate in viva), or both.

As used herein, a cell is “positive” for a particular marker when that marker is detectable. For example, a placental stem cell is positive for, e.g., CD73 because CD73 is detectable on placental stem cells in an amount detectably greater than background (in comparison to, e.g., an isotype control or an experimental negative control for any given .

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 e the cell when present or expressed by the cell.

As used herein, the term “stem cell” deﬁnes a cell that retains at least one attribute of a stem cell, e.g., a marker or gene expression proﬁle ated with one or more types of stem cells; the ability to replicate at least 10-40 times in culture; multipotency, e.g., the ability to differentiate, either in vitro, in vivo or both, into cells of one or more of the three germ layers; the lack of adult (2'.e., differentiated) cell teristics, or the like.

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

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

As used herein, the term “oligomeric or polymeric le” refers to a biomolecule that is capable of targeting a gene, RNA, or protein of interest (e.g., by binding or WO 93753 hybridizing to a region of a gene, RNA, or protein of st). This term es, for example, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, eptides or polypeptides, and any combinations (e.g., chimeric combinations) thereof. As such, these nds may be single-stranded, double-stranded, circular, branched or have ns and can comprise structural elements such as internal or terminal bulges or loops.

Oligomeric or polymeric double-stranded molecules can be two s hybridized to form double-stranded compounds or a single strand with sufﬁcient self complementarity to allow for hybridization and formation of a fully or partially double-stranded molecule.

As used herein, the term atory RNA molecule” refers to an RNA molecule that modulates, (e.g., up-regulates or down-regulates) directly or indirectly, the expression or activity of the selectable target(s) (e.g., a target gene, RNA, or protein). In certain embodiments, a atory RNA molecule” is a siRNA, miR inhibitor, miR mimic, nse RNA, shRNA, shRNAmir, or a hybrid or a combination thereof that modulates the expression of the selectable target in a host cell. In certain embodiments, the modulatory RNA molecules provided herein comprise about 1 to about 100, from about 8 to about 80, 10 to 50, 13 to 80, 13 to 50, 13 to 30, 13 to 24, 18 to 22, 19 to 23, 20 to 80, 20 to 50, 20 to 30, or 20 to 24 nucleobases (lie. from about 1 to about 100 linked nucleosides).

As used herein, the phrase “increased survival,” when describing the survival of anoikis resistant placental stem cells as compared to unmodiﬁed placental stem cells refers to the ability of the anoikis ant placental stem cells to remain viable under conditions that cause the death (e. g., by apoptosis) of unmodiﬁed placental stem cells, e.g., conditions wherein the placental stem cells cannot adhere to a substrate (e.g., a tissue culture plate or a biological substrate such as extracellular matrix) or have a diminished ability to adhere to a substrate, i.e., low-attachment conditions. In certain embodiments, increased survival of the arPSCs described herein relative to unmodiﬁed placental stem cells refers to the ability of the arPSCs to exhibit at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, ld, , 6-fold, 7-fold, 8-fold, 9- fold, or 10-fold increase in survival time when cultured under low-attachment conditions relative to an equivalent amount of unmodiﬁed placental stem cells cultured under the same conditions.

In certain embodiments, increased survival of the arPSCs described herein relative to unmodiﬁed placental stem cells refers to the ability of the arPSCs to exhibit at least a l.5-fold to ld, a 2-fold to 3-fold, a 2.5-fold to ld, a 3-fold to , a 3.5-fold to 4.5-fold, a 4-fold to 5-fold, a 5-fold to 6-fold, a 6-fold to 7-fold, a 7-fold to 8-fold, an 8-fold to , or a 9-fold to d increase in survival time when ed under low-attachment conditions relative to an equivalent amount of unmodiﬁed placental stem cells cultured under the same conditions.

Survival of arPSCs and ﬁed placental stem cells can be assessed using methods known in the art, e.g., trypan blue exclusion assay, ﬂuorescein diacetate uptake assay, propidium iodide uptake assay; thymidine uptake assay, and MTT (3-(4,5-Dimethylthiazolyl)—2,5- diphenyltetrazolium bromide) assay.

As used herein, the phrase “decreased level,” when referring to the level of expression of a given gene in an anoikis ant placental stem cell as compared to the expression of the same gene in an unmodiﬁed placental stem cell means that the expression of the gene in the anoikis resistant placental stem cell is downregulated or inhibited, resulting in, e.g., a reduction in the mRNA transcript produced by the gene and/or the protein resulting from the expression of the gene. Determination of whether or not a given gene is expressed at a decreased level can be accomplished by any art-recognized method for detection of protein production or nucleic acid production by cells, e.g. nucleic acid-based methods, e.g., northern blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), real-time PCR, quantitative PCR, and the like. Expression of proteins can be assessed using antibodies that bind to the n of interest, e.g., in an ELISA, Western blot, sandwich assay, or the like. In certain embodiments, a gene in an s resistant placental stem cell (e.g., an anoikis associated gene) is expressed at a decreased level if its sion is decreased by at least a 1.5-fold, 2-fold, 2.5- fold, 3-fold, 35-fold, 4-fold, 45-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold as compared to the expression of the gene in an unmodiﬁed placental stem cell. In n embodiments, a gene in an s resistant placental stem cell (e.g., an s associated gene) is expressed at a decreased level if its expression is decreased by at least 1.5-fold to d, 2- fold to 3-fold, 25-fold to 35-fold, 3-fold to 4-fold, 35-fold to 45-fold, 4-fold to 5-fold, 5-fold to 6-fold, 6-fold to 7-fold, 7-fold to 8-fold, 8-fold to 9-fold, or 9-fold to 10-fold as compared to the expression of the gene in an unmodiﬁed placental stem cell. 4. BRIEF DESCRIPTION OF THE FIGURES depicts growth of placental stem cells on plates that allow cell adherence (Corning Cellbind) and under low-attachment conditions on plates that do not allow cell adherence (low attachment plates: Corning Ultra-Low Attachment; Nunc Hydrocell; and Nunc Low Cell Binding). depicts microscopic images of placental stem cells es on plates that allow cell adherence (Corning Cellbind) and under low-attachment ions on a culture plate that does not allow cell adherence (Corning Ultra-Low Attachment). depicts growth of placental stem cells transduced with GFP-expressing lentiViral shRNA in tissue culture plate wells. Bright spots in the tissue plate wells correspond to GFP expression by the transduced placental stem cells. depicts the results of an MTS assay performed on placental stem cells in which speciﬁed anoikis associated genes d on the X-axis) were targeted with siRNA. Non- treated control indicates unmodiﬁed placental stem cells; NTP control indicates placental stem cells treated with non-speciﬁc siRNA. depicts the results of a CyQuant Direct Viability assay performed on placental stem cells in which speciﬁed s associated genes d on the y-aXis) were targeted with siRNA. Non-treated control indicates unmodiﬁed placental stem cells; NTP control indicates placental stem cells treated with non-speciﬁc siRNA.

FIG 6 depicts the results of a CyQuant Direct Viability assay performed on placental stem cells in which speciﬁed anoikis associated genes (listed on the ) were targeted with siRNA. NTP control indicates placental stem cells treated with non-speciﬁc siRNA.

FIG 7: cell . A) depicts growth of a population of anoikis resistant stem cells under low attachment ions (on plates that do not allow cell adherence). B) s growth of a population of unmodiﬁed placental stem cells under low attachment conditions (on plates that do not allow cell adherence).

. DETAILED DESCRIPTION .1 PRODUCTION OF ANOIKIS RESISTANT PLACENTAL STEM CELLS In one aspect, provided herein are methods of modifying placental stem cells to make them resistant to anoikis. Such methods comprise contacting the placental stem cells with an ive amount of one or more eric or ric molecules, such that one or more genes that confer anoikis in the placental stem cells is inhibited, i.e., the expression of the gene in the placental stem cells contacted with the oligomeric or polymeric molecules is lessened as ed to the expression of the gene in placental stem cells that have not been contacted with the same oligomeric or polymeric molecules. The anoikis resistant placental stem cells (arPSCs) produced by the methods described herein are placental stem cells that demonstrate an increased survival in low-attachment conditions as compared to unmodiﬁed placental stem cells. In certain embodiments, the oligomeric or polymeric les used in the methods described herein se nucleotides (e.g., DNA or RNA molecules), nucleosides, nucleotide analogs, nucleotide mimetics, polypeptides, nucleotide analogs, nucleotide mimetics, and any combinations (e.g., ic combinations) thereof.

In one embodiment, the nucleotide analog is an RNA analog, for example, an RNA analog that has been modiﬁed in the 2’-OH group, 6.g. by substitution with a group, for example -O-CH3, -O-CH2-CH2-O-CH3, -O-CH2-CH2-CH2-NH2, -CH2-CH2-OH or -F.

In certain embodiments, the oligomeric or polymeric molecules used in the methods described herein comprise one or more modiﬁcations (e.g., al ations) in the sugars, bases, or intemucleoside linkages. As used herein, the term “intemucleoside linkage group” refers to a group capable of covalently coupling together two nucleotides, such as n RNA units. Examples include phosphate, odiester groups and phosphorothioate groups. In one embodiment, the oligomeric or polymeric molecules used in the methods described herein comprise at least one phosphate intemucleoside linkage group. In one embodiment, the oligomeric or polymeric molecules used in the methods described herein comprise at least one phosphodiester intemucleoside linkage group.

In certain embodiments, the oligomeric or polymeric les used in the methods bed herein are single-stranded oligonucleotides or polynucleotides. In certain embodiments, the oligomeric or ric molecules used in the s described herein are double-stranded oligonucleotides or polynucleotides. In certain embodiments, the oligonucleotides or polynucleotides used in the s described herein comprise one or more modiﬁcations (e.g., al modiﬁcations) in the , bases, or intemucleoside linkages.

In a speciﬁc embodiment, the oligomeric molecules used in the methods described herein are modulatory RNA molecules. In certain embodiments, the modulator RNA molecules are small interfering RNAs (siRNAs), microRNA inhibitors (anti-miRs), other modulatory RNA molecules such as antisense RNAs, miR mimics, small n RNAs (shRNAs), microRNA-adapted shRNA (shRNAmirs), or any combination thereof .1.1 siRNAs In certain embodiments, the methods provided herein for the production of anoikis ant placental stem cells comprise contacting placental stem cells with an effective amount of small interfering RNAs (siRNAs), such that the resistance to anoikis in said placental stem cells is conferred, e.g., as compared to placental stem cells that have not been modiﬁed, e.g., that have not been contacted with siRNAs. As used , the term “small interfering RNA” or “siRNA” refers to an RNA molecule that interferes with the expression of a speciﬁc gene.

The siRNAs used in the methods bed herein can be single-stranded or double-stranded, and can be modiﬁed or unmodiﬁed. In one ment, the siRNAs used in the methods described herein have one or more 2'-deoxy or 2'-O-modiﬁed bases. In some embodiments, the siRNAs used in the methods described herein have one or more base substitutions and inversions (e.g., 3-4 bases ions).

In some ments, the siRNAs used in the methods described herein are double-stranded. In one embodiment, one strand of the siRNA is nse to the target nucleic acid, while the other strand is complementary to the ﬁrst strand. In certain embodiments, said siRNAs comprise a central complementary region between the ﬁrst and second strands and terminal regions that are optionally complementary between said ﬁrst and second strands or with the target RNA.

In certain embodiments, the siRNAs used in the methods described herein have a length of about 2 to about 50 nucleobases. In some embodiments, the siRNAs used in the methods described herein are double-stranded, and have a length of about 5 to 45, about 7 to 40, or about 10 to about 35 nucleobases. In some embodiments, the siRNAs used in the methods described herein are double-stranded, and are about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases in length.

In certain embodiments, one or both ends of the ﬁrst and/or second strands of the siRNAs used in the methods described herein are modiﬁed by adding one or more natural or modiﬁed nucleobases to form an overhang. In certain embodiments, one or both ends of the ﬁrst 2013/074892 and/or second strands of the siRNAs used in the methods described herein are blunt. It is possible for one end of the ﬁrst and/or second strands of the siRNAs used in the methods described herein to be blunt and the other to have overhanging nucleobases. In one embodiment, said ngs are about 1 to about 10, about 2 to about 8, about 3 to about 7, about 4 to about 6 nucleobase(s) in length. In another ment, said overhangs are about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobase(s) in length. In a speciﬁc embodiment, the siRNAs used in the s described herein are double-stranded, and have a length of about 21 nucleobases. In another speciﬁc embodiment, the siRNAs are double-stranded, and have a length of about 21 nucleobases comprising dinucleotide 3’ overhangs (e.g., dinucleotide 3' DNA ngs such as UU or TT 3'-overhangs) such that there is a 19 nt complementary region between the sense and ense strands.

In a speciﬁc embodiment, provided herein is a method of producing arPSCs, comprising contacting a placental stem cell, or population thereof, with one or more siRNAs that target one or more genes identiﬁed herein as being associated with anoikis in placental stem cells, i.e., the method comprises the ing of one or more anoikis-associated genes with one or more siRNAs. The anoikis-associated genes that can be targeted by siRNA in accordance with the methods described herein e the genes listed in Table 1, above.

In a speciﬁc embodiment, provided herein is a method of producing arPSCs, comprising contacting a placental stem cell, or population thereof, with siRNAs that target one or more of the s associated genes listed in Table 1, above. In one embodiment, said siRNAs are double-stranded. In a speciﬁc embodiment, one strand (e.g., sense ) of said double- stranded siRNAs has a sequence at least about 70%, 80%, 90%, 95%, 98% or 100% complementary to the sequence of one of the genes identiﬁed in Table 1, above (as identiﬁed based on the Gene ID of the gene provided in the table).

In another speciﬁc embodiment, the siRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene FHDCl (NCBI GENE ID NO:85462). In another speciﬁc embodiment, the siRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene GNAI2 (NCBI GENE ID NO:277l). In another speciﬁc ment, the siRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene KNDCl (NCBI GENE ID NO:85442). In another speciﬁc embodiment, the siRNAs used in the methods described herein for generating arPSCs target the tal stem cell anoikis associated gene LPAR4 (NCBI GENE ID NO:2846). In another speciﬁc embodiment, the siRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene MAP3K5 (NCBI GENE ID NO:4217). In r speciﬁc embodiment, the siRNAs used in the methods described herein for ting arPSCs target the placental stem cell anoikis associated gene SLC2A3 (NCBI GENE ID 5). In another speciﬁc embodiment, the siRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene STAU2 (NCBI GENE ID NO:27067).

In r speciﬁc embodiment, the siRNAs used in the methods bed herein for generating arPSCs target one, two, three, or more of the following placental stem cell anoikis-associated genes: FHDC1 (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID NO:2771), KNDCl (NCBI GENE ID 42), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067). In another speciﬁc embodiment, the siRNAs used in the methods described herein for generating arPSCs target one, two, three, or more of the following placental stem cell anoikis-associated genes: FHDC1 (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID NO:2771), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067), and target at least one additional anoikis associated gene recited in Table 1.

In r speciﬁc embodiment, contacting of an s-associated gene of a placental stem cell with siRNAs results in a decrease in the mRNA level of said gene in said placental stem cell, e.g., the mRNA level of the anoikis-associated gene in the resulting arPSCs is decreased relative to the mRNA level of the same gene in unmodiﬁed placental stem cells (i.e., placental stem cells not contacted with an siRNA). In certain embodiments, the mRNA level of an anoikis-associated gene in an arPSC produced according to the methods described herein is sed about, up to, or no more than, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, e.g., as compared to the expression of said gene (mRNA level) in unmodiﬁed placental stem cells.

The siRNAs used in the methods bed herein can be supplied by a commercial vendor (e.g., Ambion; Dharmacon), or be synthesized by, e.g., solid phase synthesis, or according to the procedures as described in, e.g., Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press; Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), l- 36. Gallo et al., Tetrahedron (2001), 57, 5707-5713). siRNAs useful for the production of anoikis resistant placental stem cells can be identiﬁed by a variety of methods known in the art. In certain embodiments, such siRNAs are ﬁed and obtained from one or more siRNA libraries, e.g., a commercially ble library (e. g., Ambion, Silencer® Select Human Nuclear Hormone Receptor (HNR) siRNA Library V4; Dharmacon, siRNA library Human ON-TARGETplus siRNA Nuclear Receptors Sub-Library), optionally by a screening method, e.g., medium or high-throughput screening. In one ment, such a library can encompass a wide range of genes (e.g., human -wide siRNA library), or pre-deﬁned to encompass c target genes or gene families (e.g., human nuclear receptor siRNA library, phosphatase siRNA library, etc). The screening method can be d out, for example, using ted robotics, liquid handling equipments, data processing re, and/or sensitive detectors, e.g., Precision XS Automated Pipettor System, EL406 liquid handling , or synergy plate reader. .1.2 miR inhibitors and miR mimics In certain embodiments, the methods provided herein for the production of anoikis ant placental stem cells comprise contacting placental stem cells with an effective amount of microRNA inhibitors (miR inhibitors), such that the resistance to anoikis in said placental stem cells is conferred, e.g., as compared to placental stem cells that have not been modiﬁed, e.g., that have not been contacted with miR inhibitors. As used herein, the term “microRNA,” “miRNA,” or “miR” refers to short ribonucleic acid (RNA) molecules, including, but not limited to, mature single stranded , precursor miRNAs (pre-miR), and variants thereof. As used herein, the term “microRNA inhibitor,” “miRNA inhibitor,” “miR inhibitor” or “anti-miR” refer to a ribonucleic acid le designed to inhibit miRNAs (e.g., endogenous miRNAs). In some embodiments, the miR inhibitors gulate (e. g., inhibit) a target gene by inhibition of one or more endogenous miRs. In one embodiment, the microRNAs are naturally occurring. In certain ments, the NAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs) and result in translational repression and gene ing. In certain embodiments, a single precursor contains more than one mature miRNA sequence. In other embodiments, multiple precursor miRNAs contain the same mature ce. In some embodiments, when the relative abundances y te which is the predominantly expressed miRNA, the term “microR,\IA,” ,” or “miR” refers to the predominant product, and the term “microR,\IA*,” “miRNA*,” or “miR*” refers to the opposite arm of the precursor. In one ment, miRNA is the “guide” strand that eventually enters RNA-Induced Silencing Complex (RISC), and miRNA* is the other “passenger” strand. In another embodiment, the level ofmiRNA* present in the cell at a lower level (e.g., 515%) ve to the corresponding miRNA. In some embodiments where there is a higher proportion of passenger strand present in the cell, the nomenclature miRNA-3p (i.e., miRNA derived from the 3' arm of the precursor miRNA) and miRNA-Sp (z'.e., miRNA-Sp is the miRNA derived from the 5’ arm of the sor miRNA) is used instead ofmiRNA/miRNA*.

As used herein, the term “microRNA mimic(s)” or “miR mimic(s)” refers to molecules that can be used to imitate or mimic the gene silencing ability of one or more miRNAs. In one embodiment, the miR mimics down-regulate (e.g., inhibit) a target gene by imitating one or more endogenous miRs. In certain embodiments, miRNA mimics are synthetic non-coding RNAs (2'.e., the miRNA is not obtained by puriﬁcation from a source of the endogenous miRNA). In certain embodiments, the miRNA mimics are capable of entering the RNAi pathway and ting gene expression. In certain embodiments, miRNA mimics can be designed as mature molecules (e.g. single stranded) or mimic precursors (e.g., pri- or pre- miRNAs).

In some embodiments, the miR inhibitors or miRNA mimics provided herein comprise nucleic acid (modiﬁed or modiﬁed nucleic acids) including oligonucleotides sing, e.g., RNA, DNA, modiﬁed RNA, modiﬁed DNA, locked nucleic acids, or 2'-O,4'-C- ethylene-bridged nucleic acids (ENA), or any combination of thereof.

The miR inhibitors or miR mimics can be single-stranded or double-stranded, and can be modiﬁed or unmodiﬁed. In certain embodiments, the miR tors or miR mimics have a length of about 2 to about 30 bases. In certain embodiments, the miR inhibitors or miR mimics are single-stranded, and have a length of about 15 to about 30 nucleobases. In some ments, the miR inhibitors are single-stranded, and are about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases in length.

In a speciﬁc embodiment, provided herein is a method of producing arPSCs, comprising contacting a placental stem cell, or population f, with one or more miR inhibitors or miR mimics that target one or more miRs in said placental stem cells that modulate the activity of one or more genes identiﬁed herein as being associated with anoikis in placental stem cells. The miRs that can be targeted by miR inhibitors and/or miR mimics in accordance with the methods described herein include miRs associated with the modulation of the anoikis associated genes listed in Table 1, above.

In another speciﬁc embodiment, provided herein is a method of producing arPSCs, comprising contacting a placental stem cell, or population thereof, with a miR inhibitor or miR mimic that s a miR in said tal stem cells that modulates the production of an anoikis associated gene in said tal stem cell (e.g., an anoikis associated gene listed in Table 1, above), such that the production of the s associated gene by said placental stem cells is decreased, e.g., as compared to an equivalent number of unmodiﬁed tal stem cells.

In another speciﬁc embodiment, the miR inhibitors or miR mimics used in the methods described herein for ting arPSCs target a miRNA that modulates the production of the placental stem cell anoikis associated gene FHDCl (NCBI GENE ID NO:85462). In another speciﬁc embodiment, the miR inhibitors or miR mimics used in the methods described herein for ting arPSCs target a miRNA that modulates the production of the tal stem cell anoikis associated gene GNAI2 (NCBI GENE ID NO:2771). In another speciﬁc embodiment, the miR inhibitors or miR mimics used in the methods described herein for generating arPSCs target a miRNA that modulates the production of the placental stem cell anoikis associated gene KNDCl (NCBI GENE ID 42). In r speciﬁc embodiment, the miR inhibitors or miR mimics used in the methods described herein for generating arPSCs target a miRNA that modulates the production of the placental stem cell anoikis associated gene LPAR4 (NCBI GENE ID NO:2846). In another speciﬁc embodiment, the miR tors or miR mimics used in the methods described herein for generating arPSCs target a miRNA that WO 93753 modulates the production of the placental stem cell anoikis associated gene MAP3K5 (NCBI GENE ID NO:4217). In another speciﬁc embodiment, the miR inhibitors or miR mimics used in the methods described herein for generating arPSCs target a miRNA that modulates the production of the placental stem cell anoikis associated gene SLC2A3 (NCBI GENE ID NO:6515). In another c embodiment, the miR inhibitors or miR mimics used in the methods described herein for generating arPSCs target a miRNA that modulates the production of the tal stem cell anoikis ated gene STAU2 (NCBI GENE ID NO:27067).

In another speciﬁc ment, the miR inhibitors or miR mimics used in the methods described herein for ting arPSCs target one, two, three, or more miRNAs, wherein said miRNAs modulate the production of one, two, three, or more of the following placental stem cell anoikis-associated genes: FHDCl (NCBI GENE ID 62), GNAI2 (NCBI GENE ID NO:2771), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067). In another speciﬁc embodiment, the miR inhibitors or miR mimics used in the methods described herein for generating arPSCs target one, two, three, or more miRNAs, wherein said miRNAs modulate the production of one, two, three, or more of the ing tal stem cell anoikis-associated genes: FHDCl (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID NO:2771), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067), and target at least one miRNA that modulates the production of at least one additional anoikis associated gene recited in Table 1.

In another speciﬁc embodiment, contacting ofmiRNA that modulates the production of an anoikis-associated gene of a placental stem cell with a miR inhibitor or miR mimic results in a decrease in the mRNA level of said gene in said placental stem cell, e. g., the mRVA level of the anoikis-associated gene in the resulting arPSCs is decreased relative to the mRVA level of the same gene in unmodiﬁed placental stem cells (i.e., tal stem cells not contacted with a miR inhibitor or miR mimic). In certain embodiments, the mRNA level of an anoikis-associated gene in an arPSC ed according to the methods described herein is decreased about, up to, or no more than, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, e.g., as compared to the expression of said gene (mRNA level) in unmodiﬁed placental stem cells.

The miR inhibitors and miR mimics used in the methods described herein can be supplied by a commercial vendor (e.g., Ambion; Dharmafect), or can be synthesized by, e.g., solid phase synthesis, or according to the procedures as described in, e.g., Protocols for Oligonucleotides and Analogs, Ed. l (1993), Humana Press; Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).

The miR inhibitors and miR mimics used in the s bed herein can be identiﬁed by a variety of methods known in the art. In certain embodiments, such miR inhibitors and/or miR mimics are identiﬁed and obtained from one or more miR inhibitors or miR mimics libraries, e.g., a cially available library (e.g., Ambion, Anti-miR miRNA Precursor Library Human V13), optionally by a screening method, e.g., medium or hroughput ing. In one embodiment, such a y can encompass a wide range of target miRs (e.g., human -wide siRNA library), or pre-deﬁned to encompass speciﬁc target genes or gene families (e.g., r receptor siRNA library, phosphatase siRNA library etc). The screening method can be carried out, for example, using automated robotics, liquid handling equipments, data processing re, and/or ive detectors, e.g., Precision XS Automated Pipettor System, EL406 liquid handling system, or synergy plate reader. 53WM Other modulatory RNA molecules useful for the production of arPSCs comprise antisense RNAs, shRNAs, and shRNAmirs. These RNA molecules can be used in any combination and can be used in combination with siRNAs, miR mimics and/or miR inhibitors to produce the arPSCs as described herein.

As used , the term ense RNA” is an antisense ribonucleic acid molecule. By illustration only and without limitation, the antisense RNAs hybridize to a target nucleic acid (e.g., a gene) and modulate expression activities of the target nucleic acid, such as transcription or ation.

As used herein, the term “small hairpin RNA” or “shRNA” refers to an RNA molecule comprising a stem-loop structure; the term “shRNAmir” refers to “microRNA-adapted shRNA.” In certain embodiments, said shRNA comprises a ﬁrst and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufﬁcient such that base pairing occurs between the regions, the ﬁrst and second regions being joined by a loop region, the loop resulting ﬁom a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The shRNA hairpin structure can be, for example, cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing x . This x binds to and cleaves mRNAs which match the siRNA that is bound to it.

In some embodiments, shRNAmirs or microRNA-adapted shRNA provided herein are shRNA constructs that mimic naturally occurring primary ript miRNA with the addition of an miRNA loop and a miRNA ﬂanking sequence to a shRNA. Without wishing to be bound by any theory, the shRNAmir is ﬁrst cleaved to produce shRNA by Drosha, and then cleaved again by Dicer to produce siRNA. The siRNA is then incorporated into the RISC for target mRNA ation. This allows the shRNAmir to be cleaved by Drosha thereby allowing for a r increase in knockdown efﬁciency. The addition of a miR30 loop and 125 nt of miR30 ﬂanking sequence on either side of the shRNA n has been reported to result in greater than lO-fold increase in Drosha and Dicer processing of the expressed hairpins when compared with conventional shRNA constructs without microRNA.

In a speciﬁc embodiment, provided herein is a method of producing arPSCs, comprising contacting a placental stem cell, or population thereof, with one or more antisense RNAs, , and shRNAmirs that target one or more genes identiﬁed herein as being associated with anoikis in placental stem cells, i.e., the method comprises the targeting of one or more anoikis-associated genes with one or more antisense RNAs, shRNAs, and shRNAmirs.

The anoikis-associated genes that can be targeted by antisense RNAs, , and shRNAmirs in accordance with the methods described herein include the genes listed in Table 1, above.

In another speciﬁc embodiment, the modulatory RNA les used in the methods described herein for generating arPSCs are small hairpin RNAs or shRNAs. In a speciﬁc embodiment, said shRNAs target one or more of the anoikis-associated genes listed in Table 1, above. In another speciﬁc embodiment, said shRNAs have a sequence at least about 70%, 80%, 90%, 95%, 98% or 100% complementary to the sequence of one of the genes identiﬁed in Table 1, above (as identiﬁed based on the Gene ID of the gene provided in the table).

In another embodiment, the modulatory RNA molecules used in the methods described herein for generating arPSCs are antisense RNAs. In a speciﬁc embodiment, said antisense RNAs target one or more of the anoikis-associated genes listed in Table 1, above. In another speciﬁc embodiment, said antisense RNAs have a sequence at least about 70%, 80%, 90%, 95%, 98% or 100% complementary to the sequence of one of the genes identiﬁed in Table 1, above (as identiﬁed based on the Gene ID of the gene provided in the table).

In another speciﬁc embodiment, the shRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene FHDCl (NCBI GENE ID 62). In r speciﬁc embodiment, the shRNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene GNA12 (NCBI GENE ID l). In another speciﬁc embodiment, the shRNAs used in the methods bed herein for generating arPSCs target the placental stem cell anoikis associated gene KNDCl (NCBI GENE ID NO:85442). In another speciﬁc embodiment, the shRNAs used in the methods described herein for ting arPSCs target the placental stem cell anoikis associated gene LPAR4 (NCBI GENE ID NO:2846), In another speciﬁc embodiment, the shRNAs used in the methods described herein for ting arPSCs target the placental stem cell s associated gene MAP3K5 (NCBI GENE ID NO:4217), In another speciﬁc embodiment, the shRNAs used in the methods described herein for generating arPSCs target the tal stem cell anoikis associated gene SLC2A3 (NCBI GENE ID NO:6515). In another speciﬁc embodiment, the shRNAs used in the methods described herein for ting arPSCs target the placental stem cell anoikis associated gene STAU2 (NCBI GENE ID NO:27067).

In another speciﬁc embodiment, the shRNAs used in the methods described herein for generating arPSCs target one, two, three, or more of the following placental stem cell anoikis-associated genes: FHDCl (NCBI GENE ID 62), GNA12 (NCBI GENE ID NO:277l), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAUZ (NCBI GENE ID 67). In another speciﬁc embodiment, the shRNAs used in the methods described herein for ting arPSCs target one, two, three, or more of the following placental stem cell anoikis-associated genes: FHDCl (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID NO:277l), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067), and target at least one additional anoikis ated gene recited in Table l.

In another speciﬁc embodiment, the antisense RNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene FHDCl (NCBI GENE ID NO:85462). In another speciﬁc embodiment, the antisense RNAs used in the s described herein for generating arPSCs target the placental stem cell anoikis associated gene GNAIZ (NCBI GENE ID NO:2771). In another speciﬁc embodiment, the antisense RNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene KNDCl (NCBI GENE ID NO:85442). In another speciﬁc embodiment, the nse RNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene LPAR4 (NCBI GENE ID 6). In another speciﬁc ment, the nse RNAs used in the methods described herein for generating arPSCs target the placental stem cell anoikis associated gene MAP3K5 (NCBI GENE ID NO:4217). In another speciﬁc embodiment, the antisense RNAs used in the methods described herein for ting arPSCs target the placental stem cell anoikis associated gene SLC2A3 (NCBI GENE ID NO:6515). In another speciﬁc embodiment, the antisense RNAs used in the methods described herein for ting arPSCs target the placental stem cell anoikis associated gene STAUZ (NCBI GENE ID NO:27067).

In another speciﬁc ment, the antisense RNAs used in the methods described herein for ting arPSCs target one, two, three, or more of the following placental stem cell anoikis-associated genes: FHDCl (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID NO:277l), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID 67). In another speciﬁc embodiment, the antisense RNAs used in the methods described herein for generating arPSCs target one, two, three, or more of the following placental stem cell anoikis-associated genes: FHDCl (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID NO:2771), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067), and target at least one additional anoikis associated gene recited in Table l.

In another speciﬁc embodiment, contacting of an anoikis-associated gene of a placental stem cell with an shRNA or antisense RNA results in a decrease in the mRNA level of said gene in said placental stem cell, e.g., the mRNA level of the anoikis-associated gene in the ing arPSCs is decreased relative to the mRNA level of the same gene in unmodiﬁed placental stem cells (i.e., placental stem cells not contacted with an shRNA or antisense RNA).

In n embodiments, the mRNA level of an anoikis-associated gene in an arPSC produced according to the s described herein is decreased about, up to, or no more than, 5%, 10%, %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, e.g., as compared to the expression of said gene (mRNA level) in unmodiﬁed placental stem cells.

The antisense RNAs, shRNAs and shRNAmirs used in the methods described herein can be supplied by a commercial vendor (e.g., Ambion; Dharmafect), or can be synthesized by, e.g., solid phase synthesis, or according to the procedures as described in, e.g., Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press; Scaringe, Methods (2001), 23, 206-217. Gait et (11., ations of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).

Antisense RNAs, shRNAs, shRNAmirs and other modulatory RNA les useful for the production of anoikis resistant tal stem cells can be identiﬁed by a variety of methods known in the art. In certain embodiments, such antisense RNAs, shRNAs, shRNAmirs and other modulatory RNA molecules are identiﬁed and obtained from one or more libraries, e.g., a commercially available library (Thermo Scientiﬁc, shRNAmir libraries), optionally by a screening method, e.g., medium or high-throughput screening. In one embodiment, such a library can encompass a wide range of genes (e.g., human genome targeted library), or pre- deﬁned to ass speciﬁc target genes or gene families (e.g., human nuclear receptor targeted library, atase targeted library, etc). The screening method can be carried out, for e, using automated robotics, liquid handling equipments, data processing software, and/or sensitive detectors, e.g., Precision XS Automated Pipettor , EL406 liquid ng system, or synergy plate reader.

In certain ments, the antisense RNAs, shRNAs and shRNAmirs used in the methods described herein comprise about 1 to about 100, from about 8 to about 80, 10 to 50, 13 to 80, 13 to 50, 13 to 30, 13 to 24, 18 to 22, 19 to 23, 20 to 80, 20 to 50, 20 to 30, or 20 to 24 nucleobases (nucleobases (1.6. from about 1 to about 100 linked nucleosides).

The antisense RNAs, shRNAs and shRNAmirs used in the methods described herein can be single-stranded or -stranded, modiﬁed or unmodiﬁed. In certain embodiments, said antisense RNAs, miR mimics, shRNAs, shRNAmirs and other modulatory RNA molecules comprise about 1 to about 100, from about 8 to about 80, 10 to 50, 13 to 80, 13 to 50, 13 to 30, 13 to 24, 18 to 22, 19 to 23, 20 to 80, 20 to 50, 20 to 30, or 20 to 24 nucleobases (1.6. from about 1 to about 100 linked nucleosides). In certain embodiment, the antisense RNAs, shRNAs and shRNAmirs used in the methods described herein are single-stranded, comprising from about 12 to about 35 nucleobases (1.6. from about 12 to about 35 linked nucleosides). In one embodiment, the antisense RNAs, miR mimics, shRNAs and irs used in the methods described herein are about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases in length.

The shRNAmirs used in the methods described herein can be delivered to the cells by any known method. In a speciﬁc embodiment, an shRNAmir used in the methods described herein is incorporated into a eukaryotic expression vector. In another speciﬁc embodiment, an ir used in the methods described herein is incorporated into a viral vector for gene expression. Such viral s include, but are not limited to, retroviral vectors, e. g., lentivirus, and adenoviruses. In a speciﬁc embodiment, an shRNAmir used in the methods bed herein is orated into a lentiviral vector. 54MW The tory RNA les used in the methods described herein can be delivered to placental stem cells by transfection (e.g., transient or stable transfection) or other means known in the art. In certain embodiments, said ection can be carried out, e.g., using lipids (e.g., liposomes), calcium ate, cyclodextrin, dendrimers, or polymers (e.g., cationic polymers); by electroporation, optical transfection, gene electrotransfer, impalefection, gene gun, or magnetofection; via viruses (e.g., viral carriers); or a combination thereof. In one embodiment, said transfection is performed using commercially available transfection reagents or kits (e.g., Ambion, TM Amine, siPORT NeoFX’s; Dharmafect, Dharmafect 3 Transfection Reagent or fect 1 Transfection Reagent; Invitrogen, Lipofectamine RNAiMAX; Integrated DNA Technologies, uctin; Mirus Bio LLC, TransIT-siQUEST, TransIT-TKO). In a speciﬁc embodiment, said transfection can be carried out using Dharmacon ON-TARGET plus SMARTpool® siRNA reagents with the Dharmafect l Transfection Reagent.

In some ments, said transfection can be set up in a medium or high-throughput manner, including, but not limited to, use of microtiter plate (e.g., 96-well plate) and microplate reader (e.g., synergy plate reader), or automation system, for example, ion XS Automated Pipettor System, EL406 liquid handling system. In other ments, said ection is set up in a large scale, including, but not limited to, the use of tissue e dishes or culture ﬂasks (e.g., T25, T75, or T225 ﬂasks). Placental stem cells can be plated and ed in tissue culture containers, e.g., dishes, ﬂasks, multiwell plates, or the like, for a sufﬁcient time for the placental stem cells to proliferate to about 20-80% conﬂuence, or about 30-70% nce at the time of nangbcﬁon.Forexannﬂe,ﬂuxecanbeabout2000,2500,3000,3500,or40001ﬂacmﬁalﬁen1 cells per well in a 96-well plate at the time of transfection. In one embodiment, placental stem cells are about 50% conﬂuence at the time of transfection. In another embodiment, there are about 3000 or 3500 placental stem cells per well in a 96-well plate at the time of direct transfection. In another embodiment, there are about 3500 tal stem cells per well in a 96- well plate at the time of reverse transfection.

The modulatory RNA molecules used in the methods described herein can be administered to the cells by transient transfection, or can be stably transfected to the cell for lngmmnmmmMmmagﬁmmmmmmoﬁﬁmmm“mmhmemmmbmeNAnmbwks®g, ) are ed. In one embodiment, stable transfection of modulatory RNA molecules can be d out, for example, by the use of plasmids or expression vectors that s functional modulatory RNA molecules. In one embodiment, such plasmids or expression vectors comprise a selectable marker (e.g., an antibiotic selection marker). In another embodiment, such plasmids or expression vectors comprise a cytomegalovirus (CMV) promoter, an RNA polymerase III (RNA pol III) promoter (e.g., U6 or H1), or an RNA polymerase II (RNA pol II) promoter. In another embodiment, such plasmids or expression vectors are commercially available (e.g., Ambion, pSilencerTM 4.1-CMV vector).

Other examples ofmammalian expression vectors include pLOC (Open Biosystems), which contains a cytomegalovirus promoter; pCDM8 (Seed, Nature 329:840 (1987)) and pMT2PC an et al., EMBO J. 6:187-195 (1987)). Other example expression vectors that may be used include pFNlOA (ACT) FLEXI® Vector (Promega), pFNl 1A (BIND) FLEXI® Vector (Promega), pGL4.3 1 [luc2P/GAL4UAS/Hygro] (Promega), pFC14K AG® 7) MCV FLEXI® Vector (Promega), pFClSA (HALOTAG® 7) MCV FLEXI® Vector ga), and the like.

When used in mammalian cells, an expression vector’s control functions can be provided by viral tory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus, and simian virus 40. Other suitable expression systems for both prokaryotic and eukaryotic cells are described, e.g., in rs 16 and 17 of ok et al., eds., Molecular Cloning: A Laboratog Manual, 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (1989).

Recombinant expression vectors can include one or more control sequences that can be, for example, operably linked to the nucleic acid sequence encoding the gene to be expressed. Such control sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). In certain embodiments, the vector includes a l sequence that directs constitutive expression of the tide ce in the placental stem cells. In certain other ments, the control sequence directs sion of the nucleotide sequence only in cells of certain tissues in a ent of the arPSCs, e.g., in lung, neural, muscle, skin, vascular system, or other tissues, within said recipient. In certain other embodiments, said vector comprises a control sequence that is inducible, e.g., by contact with a chemical agent, e.g., tetracycline.

The modulatory RNA les can be administered to the cells by any technique known to those of skill in the art, e.g., by direct transfection. For example, said direct transfection can involve the step of pre-plating the cells prior to transfection, allowing them to reattach and resume growth for a period of time (e.g., 24 hours) before exposure to transfection complexes. The modulatory RNA molecules can also be administered to the cells by reverse transfection. For example, said reverse transfection can involve the step of adding transfection complexes to the cells while they are in suspension, prior to plating.

In various embodiments, the effects of the modulatory RNA molecules on placental stem cells, e.g., downregulation of one or more s-associated genes in said placental stem cells so as to generate arPSCs from said placental stem cells, can last for up to, about, or no more than, 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17,18,19, 20, 21, 22 or 23lunns,or1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25, 26, 27, or 28 days, or more. In certain embodiments, the arPSCs generated using the methods described herein are used (e.g., administered to a subject) within no more than 1, 2, 3, 4, 5, 6, 7, 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22(n?23lunns,or1,2,3,4,5,6,7,8,9,10, 11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,or28(kgm(ﬁﬁhetnnethearPSCh are produced. In certain ments, the arPSCs generated using the s described herein memﬂﬂw¢egmwwmmwwﬂmmeme@giﬁﬂmmmmwmmnwameQInwmm embodiments, the effects of the modulatory RNA molecules on the arPSCs are inducible. In certain other embodiments, no, or substantially no, cellular expansion (culturing of the arPSCs, proliferation, etc.) is performed between the time the placental stem cells are modified to produce the arPSCs and the time the arPSCs are administered or cryopreserved. ment of the function (e.g., silencing of anoikis-associated genes) of the modulatory RNA molecules used in the methods described herein, e.g., the level or degree of gene silencing, can be accomplished by any art-recognized method for detection of protein production or nucleic acid production by cells. For example, assessment can be performed by ining the mRNA or protein level of a gene of st in a sample of arPSCs (e.g., a smnmeof10x105m10x107mPSCgor1%n2%h3%n4%h5%h6%h7%n8%h9%nor10%of said arPSCs) as ed to lent placental stem cells that have not been transfected or transformed with such a nucleic acid sequence. Such assessment can be performed using, e.g. nucleic acid-based methods, e.g., northern blot analysis, reverse transcriptase polymerase chain reaction R), real-time PCR, quantitative PCR, and the like. In other embodiments, expression of protein can be assessed using antibodies that bind to the protein of interest, e.g., in an ELISA, sandwich assay, or the like. In a specific embodiment, the anoikis ant placental stem cells generated using the methods described herein produce 5%, 10%, 15%, 20%, 25%, 3096,3596,4096,4596,5096,5596,6096,6596,7096,7596,8096,8596,9096,9596,or100961essof the mRNA of a target gene (e. g., an anoikis-associated gene) as ed to unmodiﬁed placental stem cells (e. g., an equivalent amount of unmodified placental stem cells (i.e., placental stem cells that have not been contacted with a modulatory RNA le). In a speciﬁc embodiment, the anoikis resistant placental stem cells generated using the methods described hereniproduce596,1096,1596,2096,2596,3096,3596,4096,4596,5096,5596,6096,6596,7096, 75%, 80%, 85%, 90%, 95%, or 100% less ofthe protein ofa target gene (e.g., an anoikis- associated gene) as compared to unmodiﬁed placental stem cells (e.g., an lent amount of ﬁed placental stem cells (i.e., placental stem cells that have not been contacted with a modulatory RNA le). .2 USES OF ANOIKIS RESISTANT PLACENTAL STEM CELLS One advantage of the arPSCs bed herein is that they maintain the functional characteristics of unmodiﬁed placental stem cells (e.g., the placental stem cells bed in US.

Patent Nos. 7,311,904; 7,311,905; 7,468,276 and 8,057,788, the disclosures ofwhich are hereby incorporated by reference in their entireties), yet are ant to anoikis and thus demonstrate sed survival in low-attachment ions as compared to, e. g., unmodiﬁed placental stem cells, which are not anoikis-resistant. Accordingly, the arPSCs described herein can be advantageously used in methods that se the administration of placental stem cells to a subject, wherein the placental stem cells are administered in a low-attachment environment, e.g., the placental stem cells are administered systemically or the placental stem cells are administered locally and do not adhere to a substrate (e.g., extracellular matrix) in the local environment.

In one embodiment, the arPSCs described herein can be used in methods of treating an individual having or at risk of developing a disease, disorder or condition caused by, or relating to, an unwanted or harmful immune response, for instance, a disease, disorder or condition having an inﬂammatory component. In another embodiment, provided herein are methods for the modulation, e.g., suppression, of the activity, e.g., proliferation, of an immune cell, or plurality of immune cells, by contacting the immune cell(s) with a plurality of arPSCs (e. g., a composition comprising arPSCs). In accordance with such methods, a therapeutically effective amount of arPSCs can be administered to the individual, wherein the stered arPSCs can e in low-attachment conditions in said individual for greater s of time than, e. g., unmodiﬁed placental stem cells administered in the same fashion.

In a speciﬁc embodiment, provided herein is a method of suppressing an immune response comprising contacting a plurality of immune cells with a plurality of anoikis resistant placental stem cells for a time sufﬁcient for said anoikis resistant placental stem cells to detectably suppress an immune response, wherein said anoikis resistant placental stem cells detectably suppress T cell proliferation in a mixed lymphocyte reaction (MLR) assay or a sion assay. An “immune cell” in the context of this method means any cell of the immune system, particularly T cells and NK al killer) cells. Thus, in various embodiments of the method, anoikis ant tal stem cells are contacted with a plurality of immune cells, wherein the plurality of immune cells are, or ses, a plurality of T cells (e.g., a ity of CD3+ T cells, CD4+ T cells and/or CD8+ T cells) and/or natural killer cells. An “immune response” in the context of the method can be any response by an immune cell to a stimulus normally perceived by an immune cell, e.g., a response to the presence of an antigen. In various ments, an immune response can be the proliferation of T cells (e.g., CD3+ T cells, CD4+ T cells 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 killer (NK) cell, the maturation of a dendritic cell, or the like. The immune response can also be a local, tissue- or organ-speciﬁc, or systemic effect of an activity of one or more classes of immune cells, e.g., the immune response can be graft versus host e, inﬂammation, formation of inﬂammation-related scar tissue, an autoimmune condition (e.g., rheumatoid arthritis, Type I diabetes, lupus erythematosus, etc). and the like. cting,” as used herein in such a context, encompasses bringing the placental stem cells and immune cells together in a single container (e.g., culture dish, ﬂask, vial, etc.) or in viva, for example, in the same individual (e.g., mammal, for example, human). In one embodiment, the contacting is for a time sufficient, and with a sufficient number of arPSCs and immune cells, that a change in an immune function of the immune cells is detectable. In certain embodiments, said contacting is sufﬁcient to suppress immune function (e.g., T cell proliferation in response to an antigen) by at least 50%, 60%, 70%, 80%, 90% or 95%, compared to the immune function in the e of the arPSCs. Such suppression in an in viva context can be determined in an in vitro assay (see below); that is, the degree of suppression in the in vitro assay can be extrapolated, for a particular number of anoikis resistant placental stem cells and a number of immune cells in a recipient dual, to a degree of suppression in the individual.

The ability of anoikis resistant tal stem cells to suppress an immune response can be, e.g., assessed in vitro. In certain embodiments, an anoikis ant placental stem cell provided herein sses an immune response at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as well as an unmodiﬁed placental stem cell (e. g., tal stem cells that are not resistant to anoikis). In certain embodiments, an anoikis resistant placental stem cell provided herein suppresses an immune response to the same extent as an unmodiﬁed placental stem cell (e. g., placental stem cells that are not resistant to anoikis). For example, a plurality of anoikis resistant placental stem cells can be tested in an MLR comprising combining CD4+ or CD8+ T cells, dendritic cells (DC) and anoikis resistant placental stem cells in a ratio of about : 1 :2, wherein the T cells are stained with a dye such as, e.g., CFSE that ions into daughter cells, and wherein the T cells are allowed to proliferate for about 6 days. The plurality of anoikis resistant placental stem cells is immunosuppressive if the T cell proliferation at 6 days in the presence of anoikis resistant placental stem cells is detectably d compared to T cell proliferation in the presence of DC and absence of placental stem cells. Additionally, a control using unmodified placental stem cells can be run in el to trate that the anoikis resistant placental stem cells are more immunosuppressive than unmodiﬁed or untreated tal stem cells. In such an MLR, for example, anoikis resistant placental stem cells can be either thawed or ted from e. About 20,000 anoikis resistant placental stem cells are resuspended in 100 ul ofmedium (RPMI 1640, 1 mM HEPES buffer, antibiotics, and 5% pooled human serum), and allowed to attach to the bottom of a well for 2 hours. CD4+ and/or CD8+ T cells are isolated from whole eral blood mononuclear cells Miltenyi magnetic beads. The cells are CFSE stained, and a total of 100,000 T cells (CD4+ T cells alone, CD8+ T cells alone, or equal amounts of CD4+ and CD8+ T cells) are added per well. The volume in the well is brought to 200 ul, and the MLR is allowed to proceed. A regression assay or BTR assay can be used in similar fashion.

In another aspect, provided herein is a method for promoting enesis. In a speciﬁc embodiment, provided herein is a method for promoting angiogenesis in a subject, e. g., a human subject, comprising administering to said subject the arPSCs described herein, or a ition f In certain embodiments, an anoikis resistant placental stem cell provided herein promotes angiogenesis at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as well as an fied placental stem cell (e. g., placental stem cells that are not resistant to anoikis). In n embodiments, an anoikis resistant placental stem cell provided herein promotes angiogenesis to the same extent as an unmodiﬁed placental stem cell (e.g., placental stem cells that are not resistant to anoikis). Assays for measuring the ability of cells (e.g., placental stem cells, including arPSCs) to promote angiogenesis are known in the art (see, e. g., US. Patent Application Publication No. 2011/0250182, the disclosure of which is herein incorporated by reference in its entirety), e.g., assaying the y of cells to promote tube formation by endothelial cells, assaying the ability of cells to promote endothelial cell migration and/or eration, and assaying the ability of cells to secrete factors that promote angiogenesis.

The anoikis resistant placental stem cells described herein can be administered with one or more second types of stem cells, e.g., mesenchymal stem cells from bone marrow.

Such second stem cells can be administered to an individual with said anoikis resistant placental stem cells in a ratio of, e. g., about 1:10 to about 10:1.

The anoikis resistant placental stem cells described herein can be administered to an individual in any manner known in the art, e.g., systemically, locally, intravenously, intramuscularly, intraperitoneally, intraocularly, parenterally, intrathecally, or directly into an organ, e.g., pancreas. For in viva stration, the anoikis resistant placental stem cells can be formulated as a pharmaceutical ition, as described below. .3 ANOIKIS RESISTANT PLACENTAL STEM CELLS AND ANOIKIS RESISTANT PLACENTAL STEM CELL POPULATIONS The anoikis resistant placental stem cells (arPSCs) provided herein are produced from placental stem cells using the s bed . In ance with the methods described herein for producing arPSCs, the arPSCs described herein express one or more anoikis-associated genes (as identiﬁed herein, e.g., one or more anoikis associated genes identiﬁed in Table 1, above) at a decreased level as ed to the expression of the same s associated gene in an unmodified tal stem cell (i.e., the expression of the one or more anoikis-associated genes is downregulated).

Placental stem cells from which anoikis ant placental stem cells are produced are not derived from blood, e.g., placental blood or umbilical cord blood. The placental stem cells used to produce the anoikis resistant tal stem cells used in the methods and compositions provided herein have the capacity, and can be selected for their capacity, to suppress the immune system of an individual.

Placental stem cells can be either fetal or maternal in origin (that is, can have the genotype of either the mother or fetus). Populations of placental stem cells, or populations of cells comprising placental stem cells, can se placental stem cells that are solely fetal or maternal in origin, or can comprise a mixed population of placental stem cells of both fetal and maternal origin. The placental stem cells, and populations of cells comprising the placental stem cells, can be identiﬁed and selected by, e.g., the morphological, marker, and culture characteristics discussed below. .3.1 al and Morphological Characteristics The placental stem cells used in the methods described herein for generating arPSCs, when cultured in primary cultures or in cell culture, adhere to the tissue culture substrate, e. g., tissue culture container surface (e.g., tissue culture plastic). Placental stem cells in culture assume a generally ﬁbroblastoid, te appearance, with a number of cytoplasmic processes extending from the central cell body. The placental stem cells used in the s for generating arPSCs described herein are, however, morphologically differentiable from ﬁbroblasts cultured under the same conditions, as the tal stem cells exhibit a greater number of such processes than do ﬁbroblasts. Morphologically, placental stem cells are also differentiable from hematopoietic stem cells, which generally assume a more rounded, or stone, morphology in culture.

The arPSCs described herein are thus distinct from, e.g., ﬁbroblasts and hematopoietic stem cells. Further, the arPSCs described herein are distinct from the tal stem cells used to generate the , ularly with respect to the y of the cells to survive in low-attachment conditions; the arPSCs described herein exhibit an increased ability to survive in low-attachment conditions relative to unmodiﬁed placental stem cells because they are resistant to anoikis, whereas the unmodiﬁed placental stem cells are not anoikis resistant. .3.2 Cell Surface, Molecular and Genetic Markers As with unmodiﬁed placental stem cells, the arPSCs described herein express a plurality of markers that can be used to identify and/or isolate the arPSCs, or populations of cells that comprise the arPSCs. Generally, the identifying s associated with the arPSCs described herein are the same as those that can be used to identify the placental stem cells from which the arPSCs are derived (i.e., the placental stem cells used in the methods described herein for ting arPSCs). Thus, the arPSCs described herein are comparable to unmodiﬁed to placental stem cells in terms of cell surface, molecular, and genetic markers, with the difference between the cells being that the arPSCs described herein express at least one of anoikis associated gene (e.g., at least one of the genes identiﬁed in Table 1, above) at a lower level relative to the expression of said gene in an equivalent amount of ﬁed placental stem 2013/074892 cells, i.e., at least one anoikis associated gene is downregulated/inhibited in the arPSCs described herein, wherein said s ated gene is not downregulated/inhibited in unmodiﬁed placental stem cells.

The arPSCs bed herein, like the placental stem cells from which the arPSCs are derived, are not bone marrow-derived mesenchymal cells, adipose-derived mesenchymal stem cells, or mesenchymal cells ed from umbilical cord blood, placental blood, or peripheral blood.

In certain embodiments, the arPSCs described herein r the placental stem cells used in the methods described herein for producing arPSCs) are CD343 CD10+ and CD105" as detected by ﬂow cytometry. In a speciﬁc ment, the isolated CD343 CDlOI, CD105" arPSCs described herein (and/or the tal stem cells used in the methods described herein for producing ) have the potential to differentiate into cells of a neural phenotype, cells of an osteogenic phenotype, and/or cells of a chondrogenic phenotype. In another speciﬁc embodiment, the isolated CD347, CDlOI, CD105+ arPSCs described herein (and/or the placental stem cells used in the methods described herein for producing arPSCs) are additionally .

In another speciﬁc embodiment, the isolated CD347, CDlOI, CD105+ arPSCs described herein (and/or the placental stem cells used in the methods described herein for producing arPSCs) are additionally CD45’ or CD90]. In another speciﬁc embodiment, the isolated CD34’, CD10: CD105+ arPSCs described herein r the placental stem cells used in the methods described herein for producing arPSCs) are additionally CD45’ and CD90: as detected by ﬂow cytometry.

In another speciﬁc ment, the isolated CD343 CD101 CDIOSI, CD200+ arPSCs bed herein (and/or the placental stem cells used in the methods described herein for producing arPSCs) are additionally CD90+ or CD45’, as detected by ﬂow cytometry. In another speciﬁc embodiment, the isolated CD347, CDlOI, CDlOSI, CD200+ arPSCs described herein (and/or the placental stem cells used in the methods described herein for producing arPSCs) are additionally CD90" and CD45’, as detected by ﬂow cytometry, z'.e., the cells are CD347, CDlOI, CD45’, CD90", CD105" and CDZOOI. In another speciﬁc ment, said CD343 CDlOI, CD45: CD90", CD105", CD200+ arPSCs described herein (and/or the placental stem cells used in the methods described herein for producing arPSCs) are additionally CD80’ and CD86’.

In certain embodiments, the arPSCs described herein (and/or the placental stem cells used in the methods described herein for producing arPSCs) are CD347, CDlOI, CD105+ WO 93753 and CD2001 and one or more of CD3 81 CD451 CD801 CD861 CD1331 HLA-DR,DP,DQ1 SSEA31 SSEA41 CD291 CD441 CD731 CD901 CD1051HLA-A,B,C1PDL11ABC-p1 and/or OCT-41 as detected by ﬂow cytometry. In other ments, any of the CD341 CD101 CD105+ arPSCs described herein (and/or the placental stem cells used in the methods described herein for producing arPSCs) are additionally one or more of CD291 CD3 81 CD441 CD541 SH3+ or SH41 In another c embodiment, the arPSCs described herein (and/or the placental stem cells used in the methods described herein for producing arPSCs) are additionally CD441 In another speciﬁc embodiment of any of the isolated CD341 CD101 CD105+ arPSCs described herein (and/or the placental stem cells used in the methods described herein for producing arPSCs) are additionally one or more of CD1 171 CD1331 KDR’ (VEGFRZ’), HLA- A,B,C1 HLA-DP,DQ,DR1 or Programmed Death-1 Ligand (PDL1)1 or any combination thereof.

In another ment, the CD341 CD101 CD105+ arPSCs described herein (and/or the placental stem cells used in the s described herein for producing arPSCs) are additionally one or more of CD131 CD291 CD331 CD381 CD441 CD451 CD541 CD62E1 CD62L1 CD62P1 SH3+ (CD73+), SH4+ (CD73+), CD801 CD861 CD901 SH2+ (CD105+), CD106/VCAM1 CD1171 VE-cadherin10w, CXCR41 CD2001 CD1331OCT-41 SSEA31 SSEA41ABC-p1 KDR’ (VEGFRZ’), HLA-A,B,C1 HLA-DP,DQ,DR1 HLA-G1 or Programmed Death-1 Ligand (PDL1)1 or any combination thereof. In another embodiment, the CD341 CD101 CD105+ arPSCs described herein (and/or the placental stem cells used in the methods described herein for producing arPSCs) are additionally CD131 CD291 CD331 CD381 CD441 CD451 CD54/ICAM1 CD62E1 CD62L1 CD62P1 SH3+ ), SH4+ (CD731, CD801 CD861 CD901 SH2+ (CD105+), CD106/VCAM1 CD1171 CD144/VE-cadherin10w, CD184/CXCR41 CD2001 CD1331 OCT-41 SSEA31 SSEA41ABC-p1KDR’(VEGFR2’), HLA-A,B,C1 HLA-DP,DQ,DR1 HLA-G1 and Programmed Death-1 Ligand (PDL1)1 In another speciﬁc embodiment, any of the arPSCs described herein (and/or the placental stem cells used in the s described herein for producing arPSCs) are additionally ABC-p1 as detected by ﬂow cytometry, or OCT-4+ (POU5F1+), as determined by reverse- riptase polymerase chain reaction R), wherein ABC-p is a placenta-speciﬁc ABC transporter protein (also known as breast cancer resistance protein (BCRP) or as mitoxantrone resistance protein (MXR)), and OCT-4 is the Octamer-4 protein l). In another speciﬁc embodiment, any of the arPSCs described herein (and/or the placental stem cells used in the methods described herein for producing arPSCs) are additionally SSEA3’ or SSEA4’, as determined by ﬂow cytometry, wherein SSEA3 is Stage Speciﬁc Embryonic n 3, and SSEA4 is Stage Speciﬁc Embryonic Antigen 4. In another speciﬁc embodiment, any of the arPSCs described herein (and/or the placental stem cells used in the methods described herein for producing arPSCs) are additionally SSEA3’ and SSEA4’.

In another speciﬁc embodiment, any of the arPSCs described herein (and/or the placental stem cells used in the methods bed herein for producing arPSCs) are, or are additionally, one or more of MHC—l+ (e.g., HLA-A,B,C+), ’ (e.g., ,DQ,DR’) or HLA-G’. In another speciﬁc embodiment, any of the arPSCs bed herein (and/or the placental stem cells used in the methods described herein for producing arPSCs) are additionally MHC—l+ (e.g., HLA-A,B,C+), MHC-II’ (e.g., HLA-DP,DQ,DR’) and HLA-G’.

Also ed herein are populations of the arPSCs described . In certain embodiments, described herein are populations of arPSCs comprising the isolated arPSCs described herein, wherein the populations of cells comprise, e.g., at least 10%, 15%, 20%, 25%, 309b,359b,409b,459b,509b,559b,609b,659b,709b,759b,809b,8596,9096,959oor989bisokued CD101 CD105+ and CD34’ arPSCs; that is, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 459b,509b,559b,609b,659b,7096,7596,809b,859b,909b,9596(M'9896tafceﬂsinsanipopulaﬁon are isolated CD101 CD105+ and CD34’ arPSCs. In a c embodiment, the isolated CD347, CD101 CD105+ arPSCs are additionally CD2001 In another speciﬁc embodiment, the isolated CD34’, CD101 CD1051 CD200+ arPSCs are additionally CD90+ or CD45’, as detected by ﬂow cytometry. In another speciﬁc embodiment, the isolated CD347, CD101 CD1051 CD200+ arPSCs are onally CD90+ and CD45’, as detected by ﬂow cytometry. In another c embodiment, any ofthe ed CD343 CD101 CD105+ arPSCs described above are additionally one or more of CD291 CD3 8’, CD441 CD541 SH3+ or SH41 In another speciﬁc embodiment, the isolated CD343 CD101 CD105+ arPSCs, or isolated CD343 CD101 CD1051 CD200+ placental stem cells, are additionally CD441 In a speciﬁc embodiment of any of the populations of cells comprising ed CD347, CD101 CD105+ arPSCs above, the isolated arPSCs are additionally one or more of CD131 CD291 CD331 CD387, CD441 CD457, CD541 CD62E’, CD62L’, CD621): SH3+ (CD73+), SH4+ (CD73+), CD80: CD86: CD901 SH2+ (CD105+), CD106/VCAM1 CD117’, CD144/VE-cadherin10w, CD184/CXCR4’, CD2001 WO 93753 CD1331 OCT-41 SSEA3’, SSEA4’, ABC—p1 KDR’ (VEGFRZ’), HLA—A,B,C1 HLA- DR’, HLA-G’, or mmed Death-l Ligand (PDLl)+, or any combination thereof. In r speciﬁc embodiment, the CD34’, CD10: CD105+ arPSCs are additionally CD13", CD291 CD33", CD38: CD441 CD451 CD54/1CAM1 CD62E’, CD62L’, CD621): 5H3" (CD73+), 8H4" (CD73+), CD80: CD86: CD901 SH2+ (CD105+), CD106/VCAM1 CD117: CD144/VE—cadherin10W, CD184/CXCR41 CD2001 CD1331 OCT—41 SSEA3’, , ABC- p1 KDR’ (VEGFRZ’), HLA—A,B,C1HLA—DP,DQ,DR1 HLA-G’, and Programmed Death-l Ligand (PDL1)1 In certain embodiments, the isolated arPSCs in said population of cells are one or more, or all, of CD101 CD291 CD341 CD381 CD441 CD451 CD541 CD901 SH21 SH31 SH4+, SSEA3’, SSEA4’, OCT-4+, and ABC-pt wherein said the placental stem cells used in the method of generating said isolated arPSCs were obtained by physical and/or enzymatic disruption of placental tissue. In a speciﬁc ment, the isolated arPSCs are OCT-4+ and ABC-pi In another speciﬁc embodiment, the isolated arPSCs are OCT-4+ and CD34’, wherein said isolated arPSCs have at least one of the following characteristics: CD10: CD29: CD44: CD45: CD541 CD901 SH31 SH41 SSEA3’, and SSEA4’. In another speciﬁc embodiment, the isolated arPSCs are OCT—41 CD341 CD101 CD291 CD441 CD451 CD541CD901 SH31 SH4+, , and SSEA4’. In another embodiment, the isolated arPSCs are OCT-4+, CD347, SSEA3’, and SSEA4’. In another speciﬁc ment, the isolated arPSCs are OCT-4+ and CD34’, and is either SH2+ or SH3+. In another speciﬁc embodiment, the isolated arPSCs are OCT-4", CD347, SH2+, and SH3+. In r speciﬁc embodiment, the isolated arPSCs are OCT-4", CD347, SSEA3’, and SSEA4’, and are either SH2+ or SH3+. In another speciﬁc embodiment, the isolated arPSCs are OCT-4+ and CD34’, and either SH2+ or SH3+, and at least one of CD101 CD291 CD441 CD451 CD541 CD901 SSEA3’, or SSEA4’. In another speciﬁc ment, the isolated arPSCs are OCT—41 CD341 CD101 CD291 CD441 CD451 CD541 CD901 , and SSEA4’, and either SH2+ or SH31 In another ment, the isolated arPSCs are SH2+, SH3+, SH4+ and OCT-4+.

In another speciﬁc embodiment, the isolated arPSCs are CDlOl, CD29: CD44: CD54: CD90: CD34’, CD45’, SSEA3’, or SSEA4’. In another embodiment, the isolated arPSCs are SH2+, SH3+, SH4+, SSEA3’ and SSEA4’. In another speciﬁc embodiment, the isolated arPSCs are WO 93753 SH21 SH31 SH41 SSEA3’ and SSEA4", CD101 CD291 CD441 CD541 CD901 OCT-41 CD34’ or CD45’.

In another embodiment, the isolated arPSCs described herein are CDlO+, CD29+, CD34, CD441 CD451 CD541 CD901 SH21 SH31 and SH41 wherein said isolated arPSCs are additionally one or more of OCT-4+, SSEA3’ or SSEA4’.

In certain embodiments, isolated arPSCs are CD200+ or HLA-G’. In a speciﬁc embodiment, the isolated arPSCs are CD200+ and HLA-G’. In another speciﬁc embodiment, the isolated arPSCs are additionally CD73+ and CD105+. In another speciﬁc embodiment, the isolated arPSCs are additionally CD347, CD3 8’ or CD45’. In r speciﬁc ment, the isolated arPSCs are onally CD347, CD3 8’ and CD45’. In another speciﬁc embodiment, said arPSCs are CD341 CD381 CD451 CD73+ and CD1051 In another c embodiment, said isolated CD200+ or HLA-G’ arPSCs facilitate the formation of id-like bodies in a population of placental cells comprising the isolated placental stem cells, under conditions that allow the formation of embryoid-like . In another speciﬁc embodiment, the isolated arPSCs are isolated away from placental cells that are not said arPSCs. In another speciﬁc embodiment, said isolated arPSCs are isolated away from placental cells that do not display this combination of s.

In another embodiment, a cell population useful in the methods and compositions described herein is a population of cells comprising, e.g., that is enriched for, , HLA-G’ arPSCs. In a speciﬁc embodiment, said population is a population of tal cells. In s embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of cells in said cell population are isolated CD200+, HLA- G’ arPSCs. Preferably, at least about 70% of cells in said cell population are isolated CD200+, HLA-G’ arPSCs. More preferably, at least about 90%, 95%, or 99% of said cells are isolated CD200", HLA-G’ arPSCs. In a speciﬁc embodiment of the cell populations, said isolated CD200", HLA-G’ arPSCs are also CD73+ and CD105+. In another speciﬁc embodiment, said isolated CD200: HLA-G’ arPSCs are also CD347, CD3 8’ or CD45’. In another speciﬁc embodiment, said isolated , HLA-G’ arPSCs are also CD347, CD3 8’, CD45’, CD73+ and CD105+. In another embodiment, said cell population produces one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies. In another speciﬁc embodiment, said cell population is isolated away from placental cells that are not arPSCs. In another speciﬁc embodiment, said isolated CD200: HLA-G’ arPSCs are isolated away from placental cells that do not display these markers.

In another embodiment, the ed arPSCs described herein are CD73: CD105: and CD200: In another speciﬁc embodiment, the ed arPSCs are HLA-G’. In another speciﬁc embodiment, the isolated arPSCs are CD34’, CD3 8’ or CD45’. In r speciﬁc embodiment, the isolated arPSCs are CD347, CD3 8’ and CD45’. In another speciﬁc ment, the isolated arPSCs are CD347, CD3 8’, CD45’, and HLA-G’. In another speciﬁc ment, the isolated CD73: CD105: and CD200+ arPSCs facilitate the formation of one or more embryoid-like bodies in a population of placental cells comprising the isolated arPSCs, when the population is cultured under conditions that allow the formation of embryoid-like bodies. In another speciﬁc ment, the isolated arPSCs are isolated away from placental cells that are not the isolated arPSCs. In another speciﬁc embodiment, the isolated arPSCs are isolated away from placental cells that do not display these markers.

In another embodiment, a cell population useful in the s and compositions bed herein is a population of cells comprising, e.g., that is enriched for, ed CD73: CD105: CD200+ arPSCs. In various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of cells in said cell population are isolated CD73: CD105: CD200+ arPSCs. In another embodiment, at least about 70% of said cells in said population of cells are isolated CD73: CD105: CD200+ arPSCs. In r embodiment, at least about 90%, 95% or 99% of cells in said population of cells are isolated CD73: CD105: CD200+ arPSCs. In a speciﬁc ment of said populations, the isolated arPSCs are HLA-G’. In another speciﬁc embodiment, the isolated arPSCs are onally CD347, CD3 8’ or CD45’. In another speciﬁc embodiment, the isolated arPSCs are additionally CD347, CD3 8’ and CD45’. In another speciﬁc embodiment, the isolated arPSCs are additionally CD347, CD3 8’, CD45’, and HLA-G’. In another speciﬁc embodiment, said population of cells produces one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies. In another speciﬁc embodiment, said population of arPSCs is isolated away from placental cells that are not arPSCs. In another speciﬁc embodiment, said population of arPSCs is isolated away from tal cells that do not display these characteristics.

In n other embodiments, the isolated arPSCs are one or more of CD10: CD291 CD341CD381CD441 CD451 CD541 CD901 SH21 SH3: SH4: , SSEA4’, OCT-4: HLA-G’ or ABC-p: In a speciﬁc ment, the isolated arPSCs are CDlO: CD29+ u CD34: CD38: CD441 CD451 CD541 CD901 SH2 1 SH3 1 SH4 1 SSEA3-, SSEA4’, and OCT-4: In another speciﬁc embodiment, the isolated arPSCs are CD10: CD29: CD34’, CD38’, CD45’, CD54: SH2: SH3: and SH4: In another c embodiment, the ed arPSCs CD101 CD291 CD341 CD381CD451CD541 SH2: SH3: SH4+ and OCT—41 In another speciﬁc embodiment, the isolated arPSCs are CD10: CD29: CD34’, CD38’, CD44: CD45’, CD54: CD90: HLA-G’, SH2: SH3: SH4: In another speciﬁc embodiment, the isolated arPSCs are OCT-4+ and ABC-p: In another speciﬁc embodiment, the isolated arPSCs are SH2: SH3: SH4+ and OCT-4: In another embodiment, the isolated arPSCs are OCT-4: CD341 SSEA3’, and SSEA4’. In a speciﬁc ment, said isolated OCT-4: CD347, SSEA3’ and SSEA4’ arPSCs are additionally CD101 CD291 CD341 CD441 CD451 CD541 CD901 SH2: SH3: and SH4: In another embodiment, the ed arPSCs are OCT-4+ and CD34’, and either SH3+ or SH4: In another embodiment, the isolated arPSCs are CD34’ and either CD101 CD291 CD441 CD541 CD901 or OCT—41 In another embodiment, isolated arPSCs are CD200+ and OCT-4: In a speciﬁc embodiment, the isolated arPSCs are CD73+ and CD105: In another speciﬁc embodiment, said isolated arPSCs are HLA-G’. In another speciﬁc embodiment, said isolated CD200: OCT-4+ arPSCs are CD341 CD3 8’ or CD45: In another speciﬁc embodiment, said isolated CD200: OCT-4" arPSCs are CD347, CD3 8’ and CD45: In another speciﬁc embodiment, said ed CD200", OCT-4+ arPSCs are CD341 CD381 CD45, CD73: CD105+ and HLA-G’. In another speciﬁc embodiment, the ed CD200: OCT-4+ arPSCs facilitate the production of one or more embryoid-like bodies by a population of placental cells that comprises the arPSCs, when the population is cultured under conditions that allow the formation of id-like bodies. In another speciﬁc embodiment, said isolated CD200: OCT-4+ arPSCs are isolated away from placental cells that are not said arPSCs. In another speciﬁc embodiment, said isolated CD200: OCT-4+ arPSCs are isolated away from placental cells that do not display these characteristics.

In another embodiment, a cell population useful in the methods and compositions described herein is a population of cells comprising, e.g., that is enriched for, CD200: OCT-4+ arPSCs. In various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of cells in said cell population are isolated CD200: OCT-4+ . In another embodiment, at least about 70% of said cells are said isolated CD200: OCT-4+ arPSCs. In another embodiment, at least about 80%, 90%, 95%, or 99% of cells in said cell population are said isolated CD200: OCT-4+ arPSCs. In a speciﬁc embodiment of the isolated populations, said isolated CD200: OCT-4+ arPSCs are additionally CD73+ and CD105: In another speciﬁc embodiment, said isolated CD200: OCT-4+ arPSCs are onally HLA-G’. In another speciﬁc embodiment, said isolated CD200: OCT-4+ arPSCs are additionally CD347, CD3 8’ and CD45’. In another speciﬁc embodiment, said isolated CD200: OCT-4+ arPSCs are additionally CD343 CD3 8’, CD45: CD73: CD105+ and HLA-G’.

In another speciﬁc embodiment, the cell population es one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies. In another c embodiment, said cell population is isolated away from placental cells that are not isolated CD200: OCT-4+ arPSCs. In r speciﬁc embodiment, said cell population is isolated away from placental cells that do not display these markers.

In another embodiment, the isolated arPSCs useful in the methods and compositions described herein are CD73: CD105+ and HLA-G’. In another speciﬁc embodiment, the isolated CD73: CD105+ and HLA-G’ arPSCs are additionally CD347, CD3 8’ or CD45’. In another speciﬁc embodiment, the isolated CD73: CD105: HLA-G’ arPSCs are additionally CD34: CD3 8’ and CD45’. In another speciﬁc embodiment, the isolated CD73: CD105: HLA-G’ arPSCs are additionally OCT-4: In r speciﬁc embodiment, the isolated CD73: CD105: HLA-G’ arPSCs are additionally CD200: In r speciﬁc embodiment, the isolated CD73: CD105: HLA-G’ arPSCs are additionally CD343 CD3 8’, CD45; OCT-4+ and CD200: In another speciﬁc embodiment, the isolated CD73: CD105: HLA-G’ arPSCs facilitate the formation of embryoid-like bodies in a population of placental cells comprising said , when the tion is cultured under conditions that allow the formation of embryoid- like bodies. In another speciﬁc embodiment, the ed CD73: CD105: HLA-G’ arPSCs are isolated away from tal cells that are not the isolated CD73: CD105: HLA-G’ arPSCs. In another speciﬁc embodiment, said the isolated CD73: CD105: HLA-G’ arPSCs are isolated away from placental cells that do not display these s.

In another embodiment, a cell tion useful in the methods and compositions described herein is a population of cells comprising, e.g., that is enriched for, isolated CD73: CD105+ and HLA-G’ arPSCs. In various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of cells in said population of cells are isolated CD73+, CD105+, HLA-G’ arPSCs. In another embodiment, at least about 70% of cells in said population of cells are isolated CD73+, CD105+, HLA-G’ arPSCs. In another ment, at least about 90%, 95% or 99% of cells in said population of cells are ed CD73+, CD105+, HLA-G’ arPSCs. In a speciﬁc embodiment of the above populations, said isolated CD73+, CD105+, HLA-G’ arPSCs are additionally CD347, CD3 8’ or CD45’. In another speciﬁc embodiment, said isolated CD73+, CD105+, HLA-G’ arPSCs are additionally CD347, CD3 8’ and CD45’. In another speciﬁc embodiment, said ed CD73+, CD105+, HLA-G’ arPSCs are additionally OCT-4+. In another speciﬁc embodiment, said isolated CD73+, CD105+, HLA-G’ arPSCs are additionally CD200+. In another c ment, said ed CD731 CDlosi HLA-G’ arPSCs are additionally CD343 CD3 8’, CD45’, OCT-4+ and CD200+. In another speciﬁc embodiment, said cell population is isolated away from placental cells that are not CD73+, CD105+, HLA-G’ arPSCs. In another speciﬁc embodiment, said cell population is ed away from placental cells that do not display these markers.

In another embodiment, the isolated arPSCs are CD73+ and CD105+ and facilitate the formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said CD73+, CD105+ cells when said population is cultured under conditions that allow formation of embryoid-like bodies. In another c embodiment, said isolated CD73+, CD105+ arPSCs are additionally CD347, CD3 8’ or CD45’. In another speciﬁc embodiment, said isolated CD73+, CD105+ arPSCs are onally CD347, CD3 8’ and CD45’. In another speciﬁc embodiment, said isolated CD73", CD105" arPSCs are additionally OCT-4". In another speciﬁc embodiment, said ed CD73", CD105" arPSCs are additionally OCT-4", CD347, CD3 8’ and CD45’. In r speciﬁc embodiment, said isolated CD73+, CD105+ arPSCs are isolated away from placental cells that are not said cells. In another speciﬁc embodiment, said isolated CD73+, CD105+ arPSCs are isolated away from placental cells that do not display these characteristics.

In another embodiment, a cell population useful in the methods and compositions described herein is a population of cells comprising, e.g., that is enriched for, isolated arPSCs that are CD73+, CD105+ and facilitate the formation of one or more id-like bodies in a population of ed tal cells comprising said cells when said population is cultured under conditions that allow formation of embryoid-like bodies. In various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of cells in said population of cells are said isolated CD73: CD105+ arPSCs. In another embodiment, at least about 70% of cells in said population of cells are said isolated CD73: CD105+ arPSCs. In another ment, at least about 90%, 95% or 99% of cells in said population of cells are said isolated CD73: CD105+ arPSCs. In a c embodiment of the above populations, said isolated CD73: CD105+ arPSCs are additionally CD343 CD3 8’ or CD45’. In another speciﬁc embodiment, said isolated CD73: CD105+ arPSCs are additionally CD343 CD3 8’ and CD45’. In another speciﬁc embodiment, said isolated CD73: CD105+ arPSCs are additionally OCT-4". In another speciﬁc embodiment, said isolated CD73", CD105" arPSCs are additionally CD200". In another speciﬁc embodiment, said isolated CD73", CD105" arPSCs are additionally CD347, CD3 8’, CD45’, OCT-4+ and CD200+. In another speciﬁc embodiment, said cell population is isolated away from placental cells that are not said isolated CD73: CD105+ arPSCs. In another speciﬁc embodiment, said cell population is isolated away from placental cells that do not display these markers.

In another embodiment, the isolated arPSCs are OCT-4+ and facilitate formation of one or more id-like bodies in a population of isolated placental cells comprising said arPSCs when said population of cells is cultured under conditions that allow formation of embryoid-like bodies. In a speciﬁc embodiment, said isolated OCT-4+ arPSCs are additionally CD73+ and CD105+. In r c ment, said isolated OCT-4+ arPSCs are additionally CD347, CD3 8’, or CD45’. In another speciﬁc embodiment, said isolated OCT-4+ arPSCs are onally CD200". In r speciﬁc embodiment, said isolated OCT-4+ arPSCs are additionally CD731 CD105", CD200+, CD343 CD3 8’, and CD45. In another speciﬁc embodiment, said isolated OCT-4+ arPSCs are isolated away from placental cells that are not OCT-4+ arPSCs. In another speciﬁc embodiment, said isolated OCT-4+ arPSCs are isolated away from placental cells that do not display these characteristics.

In another embodiment, a cell population useful in the methods and itions described herein is a tion of cells comprising, e.g., that is enriched for, isolated arPSCs that are OCT-4+ and facilitate the formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said cells when said tion is cultured under conditions that allow ion of embryoid-like bodies. In various embodiments, at least about %, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of cells in said population of cells are said isolated OCT-4+ arPSCs. In another embodiment, at least about 70% of cells in said population of cells are said isolated OCT-4+ arPSCs. In another ment, at least about 80%, 90%, 95% or 99% of cells in said population of cells are said isolated OCT-4+ . In a speciﬁc embodiment of the above populations, said isolated OCT-4+ arPSCs are additionally CD347, CD3 8’ or CD45’. In another speciﬁc embodiment, said isolated OCT-4+ arPSCs are additionally CD34’, CD3 8’ and CD45’.

In another speciﬁc embodiment, said isolated OCT-4+ arPSCs are additionally CD73+ and CD105". In another speciﬁc embodiment, said isolated OCT-4" arPSCs are additionally CD200". In another speciﬁc embodiment, said isolated OCT-4" arPSCs are additionally CD73+, CD105", CD200+, CD34’, CD38’, and CD45’. In another speciﬁc embodiment, said cell population is isolated away from placental cells that are not said arPSCs. In another speciﬁc embodiment, said cell tion is isolated away from tal cells that do not display these In another embodiment, the isolated placental stem cells useful in the methods and compositions described herein are isolated HLA-A,B,C+, CD45’, CD133’ and CD34’ arPSCs. In another embodiment, a cell population useful in the methods and itions described herein is a population of cells comprising isolated arPSCs, wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of cells in said population of cells are isolated HLA-A,B,C+, CD457, CD133’ and CD34’ arPSCs. In a speciﬁc embodiment, said isolated arPSCs or population of isolated arPSCs is isolated away from placental cells that are not HLA-A,B,C+, CD457, CD133’ and CD34’ arPSCs. In another speciﬁc embodiment, said isolated arPSCs are non-matemal in origin. In another speciﬁc embodiment, said population of isolated arPSCs are ntially free of maternal components; e.g., at least about 40%, 45%, 5-0%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells in said population of isolated arPSCs are non-matemal in origin.

In another embodiment, the isolated arPSCs useful in the methods and compositions described herein are ed CD 10+, CD 1 3+, CD33+, CD45’, CD1 17’ and CD 1 33’ arPSCs. In another ment, a cell population useful in the s and compositions described herein is a population of cells comprising ed , wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of cells in said 2013/074892 population of cells are isolated CD10+, CD13: CD33: CD45’, CD117’ and CD133’ arPSCs. In a speciﬁc embodiment, said isolated arPSCs or population of isolated arPSCs is isolated away from placental cells that are not said isolated arPSCs. In another speciﬁc embodiment, said isolated CD10+, CD13: CD33: CD45’, CD117’ and CD133’ arPSCs are non-matemal in origin, z'.e., have the fetal genotype. In another speciﬁc embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells in said population of ed arPSCs, are non-matemal in . In another speciﬁc embodiment, said ed arPSCs or population of isolated arPSCs are isolated away from tal cells that do not display these characteristics.

In another ment, the isolated arPSCs are isolated CD10+ CD33’, CD44: CD45’, and CD117’ arPSCs. In another embodiment, a cell population useful for the in the methods and compositions described herein is a population of cells comprising, e.g., enriched for, isolated arPSCs, wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of cells in said population of cells are ed CD10+ CD33’, CD44+, CD45’, and CD117’ arPSCs. In a speciﬁc embodiment, said isolated arPSCs or population of isolated arPSCs is isolated away from placental cells that are not said cells. In another speciﬁc embodiment, said isolated arPSCs are non-matemal in origin. In another speciﬁc embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said arPSCs in said cell population are non-matemal in origin. In another speciﬁc embodiment, said isolated arPSCs or population of isolated arPSCs is ed away from placental cells that do not display these markers.

In another embodiment, the isolated arPSCs useful in the s and compositions described herein are isolated CD10+ CD13’, CD33’, CD45’, and CD117’ arPSCs.

In another embodiment, a cell population useful in the methods and compositions described herein is a population of cells comprising, e. g., ed for, ed CD10+, CD13’, CD33’, CD45’, and CD117’ arPSCs, wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of cells in said population are CD10+ CD13’, CD33’, CD45’, and CD117’ arPSCs. In a speciﬁc embodiment, said isolated arPSCs or population of ed arPSCs are isolated away from placental cells that are not said arPSCs. In another speciﬁc embodiment, said isolated placental cells are non-matemal in origin. In another speciﬁc embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells in said cell population are non-matemal in origin. In another speciﬁc embodiment, said isolated arPSCs or population of isolated arPSCs is isolated away from placental cells that do not display these characteristics.

In another embodiment, the isolated arPSCs useful in the methods and compositions described herein are HLA A,B,C+, CD45’, CD34’, and CD133’, and are additionally CD101 CD131 CD381 CD441 CD901 CD1051 CD200+ and/or HLA-G’, and/or negative for CD117. In another embodiment, a cell population useful in the methods described herein is a population of cells sing isolated arPSCs, wherein at least about 20%, 25%, 3096,3596,4096,4596,5096,5596,6096,6596,7096,7596,8096,8596,9096,9596,9896(n?about 99% of the cells in said population are isolated arPSCs that are HLA A,B,C’, CD45’, CD34’, CD133’, and that are additionally positive for CD10, CD13, CD38, CD44, CD90, CD105, CD200, and/or negative for CD117 and/or HLA-G. In a speciﬁc embodiment, said isolated arPSCs or population of isolated arPSCs are ed away from placental cells that are not said arPSCs. In r speciﬁc embodiment, said ed arPSCs are non-matemal in origin. In another speciﬁc embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said arPSCs in said cell population are non-matemal in origin.

In another speciﬁc embodiment, said isolated arPSCs or population of ed arPSCs are isolated away from placental cells that do not display these characteristics.

In r embodiment, the isolated arPSCs are isolated arPSCs that are CD200+ and CD10+, as determined by antibody g, and CD117’, as determined by both dy g and RT-PCR. In another embodiment, the isolated arPSCs are isolated placental stem cells that are CD10+, CD297, CD54+, CD200+, HLA-G’, MHC class I+ and Bmicroglobulinl.

In another embodiment, isolated arPSCs useful in the methods and compositions described herein are arPSCs wherein the expression of at least one cellular marker is at least two-fold higher than in an equivalent number of mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stem cells. In another c embodiment, said isolated arPSCs are non-matemal in origin. In another speciﬁc embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells in said cell population are non-matemal in origin.

In another embodiment, the isolated arPSCs are isolated arPSCs that are one or more of CD101 CD291 CD441 CD451 CD54/1CAM1 CD62E’, CD62L1CD62P1 CD80, CD867, CD103’, CD104’, CD105+, CD106/VCAM+, CD144/VE-cadherin10w, CD184/CXCR4’, B2-microglobulin10w, MHC-Ilow, MHC-II’, HLA-Glow, and/or W. In a speciﬁc embodiment, the isolated arPSCs are at least CD29" and CD54+. In another speciﬁc embodiment, the isolated arPSCs are at least CD44" and CD106+. In another c embodiment, the isolated arPSCs are at least CD29".

In another embodiment, a cell population useful in the methods and compositions described herein comprises isolated arPSCs, and at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cells in said cell population are isolated arPSCs that are one or more of CD10+, CD291 CD441CD451 CD54/1CAM1CD62—E1 CD62-L1 CD62-P1 CD801 CD861 CD1031 CD104: CD1051 CD106/VCAM1 CD144/VE-cadherindim, CD184/CXCR41 [32— microglobulindim, HLA-Idim, HLA-II’, HLA-Gdim, and/or m arPSCs. In another speciﬁc embodiment, at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of cells in said cell population are CD101 CD291 CD441 CD451 CD54/1CAM1 CD62—E1 CD62—L1 CD62-P’, CD80: CD86: CD103: CD104: CD1051CD106NCAM1 CD144/VE-cadherindim, CD184/CXCR41 BZ-microglobulindim, MHC—Idim, MHC-II’, HLA-Gdim, and 13DL1dim . In n embodiments, the arPSCs express HLA-II markers when induced by interferon gamma In another embodiment, the isolated arPSCs useful in the methods and compositions described herein are isolated arPSCs that are one or more, or all, of CD10+, CD29+, CD34: CD38: CD441 CD451 CD541 CD901 SH2 1 SH3 1 SH4 1 SSEA3’, SSEA4’, OCT—41 and ABC-pl, where ABC-p is a placenta-speciﬁc ABC transporter protein (also known as breast cancer resistance protein (BCRP) or as mitoxantrone resistance protein (MXR)), n said ed arPSCs are derived from placental stem cells obtained by perﬁlsion of a mammalian, e. g., human, placenta that has been drained of cord blood and perfused to remove residual blood.

In r speciﬁc embodiment of any of the above embodiments, sion of the recited cellular marker(s) (e.g., cluster of differentiation or immunogenic marker(s)) is determined by ﬂow cytometry. In another speciﬁc embodiment, expression of the marker(s) is determined by RT-PCR.

Gene ng conﬁrms that isolated , and populations of isolated arPSCs, are distinguishable from other cells, e.g., mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stem cells. The isolated arPSCs described herein can be distinguished from, e.g., bone marrow-derived mesenchymal stem cells on the basis of the expression of one or more genes, the expression of which is signiﬁcantly higher in the isolated arPSCs in comparison to bone marrow-derived mesenchymal stem cells. In ular, the isolated arPSCs, useful in the methods of treatment provided , can be distinguished from bone -derived mesenchymal stem cells on the basis of the expression of one or more genes, the expression of which is signiﬁcantly higher (that is, at least twofold higher) in the isolated arPSCs than in an equivalent number of bone -derived mesenchymal stem cells, wherein the one or more gene se ACTG2, ADARBl, AMIGO2, ARTS-l, B4GALT6, BCHE, Cl lorf9, CD200, COL4Al, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RLl, FLJ1078l, GATA6, GPR126, GPRCSB, ICAMl, IER3, IGFBP7, ILlA, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST,NFE2L3,NUAK1, PCDH7, PDLIM3, PKP2, RTNl, SERPINB9, ST3GAL6, ST6GALNAC5, SLCl2A8, TCF2l, TGFB2, VTN, ZC3Hl2A, or a combination of any of the foregoing, when the cells are grown under equivalent conditions. See, e. g., US. Patent Application Publication No. 2007/0275362, the disclosure of which is incorporated herein by reference in its entirety. In n specific embodiments, said expression of said one or more genes is determined, e.g., by RT-PCR or microarray analysis, e.g., using a Ul33-A microarray (Affymetrix).

In another specific embodiment, said isolated arPSCs express said one or more genes when cultured for a number of population doublings, e.g., anywhere from about 3 to about population ngs, in a medium comprising DMEM-LG (e.g., from Gibco); 2% fetal calf serum (e.g., from Hyclone Labs.); lx insulin-transferrin-selenium (ITS); lx linoleic acid-bovine serum albumin (LA-BSA); 10'9 M dexamethasone (e.g., from Sigma); 10'4 M ascorbic acid 2- phosphate (e.g., from Sigma); mal growth factor 10 ng/mL (e.g., from R&D Systems); and platelet-derived growth factor (PDGF-BB) 10 ng/mL (e.g., from R&D Systems). In another specific embodiment, the placental cell-specific gene is CD200.

Specific sequences for these genes can be found in GenBank at accession nos.

NM_001615 (ACTG2), BC065545 l), (NM_l 8 l 847 2), AY358590 (ARTS- l), BC074884 (B4GALT6), BC008396 (BCHE), 96 (Cl lorf9), BC03 l 103 (CD200), NM_001845 (COL4Al), NM_001846 (COL4A2), BC052289 (CPA4), BC094758 (DMD), AF293359 (DSC3), NM_001943 (DSG2), AF338241 (ELOVL2), AY336105 (F2RLl), NM_018215 (FLJ1078l), AY4l6799 (GATA6), BC075798 6), 235 (GPRCSB), AF340038 (ICAMl), BC000844 (IER3), BC066339 (IGFBP7), BC013 142 (ILlA), BT019749 (IL6), BC00746l (ILl 8), 017) KRT l 8, BC075 839 (KRT8), BC060825 (LIPG), BC065240 (LRAP), BC010444 (MATN2), BC011908 (MEST), BC068455 (NFE2L3), NM_014840 (NUAKl ), AB006755 (PCDH7), NM_014476 (PDLIM3), BC 126199 (PKP-2), BC090862 (RTNl), BC002538 (SERPINB9), BC0233 12 (ST3GAL6), BC001201 (ST6GALNAC5), BC 126 l 60 or BC065328 (SLC l2A8), BC025697 (TCF21), BC096235 (TGFB2), 46 (VTN), and 01 (ZC3Hl2A) as of March 2008.

In certain speciﬁc embodiments, said isolated arPSCs express each ofACTG2, ADARBl, AMIGO2, ARTS-l, B4GALT6, BCHE, Cl lorﬁ), CD200, COL4Al, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RLl, FLJ1078l, GATA6, GPR126, , ICAMl, IER3, , ILlA, IL6, IL18, KRTl8, KRT8, LIPG, LRAP, MATN2, MEST, ,NUAK1, PCDH7, PDLIM3, PKP2, RTNl, SERPINB9, ST3GAL6, ST6GALNAC5, SLCl2A8, TCF21, TGFB2, VTN, and ZC3Hl2A at a detectably higher level than an lent number ofbone marrow-derived mesenchymal stem cells, when the cells are grown under equivalent conditions.

In speciﬁc embodiments, the arPSCs express CD200 and ARTSl (aminopeptidase regulator of type 1 tumor necrosis factor); ARTS-l and LRAP cyte-derived arginine aminopeptidase); IL6 leukin-6) and TGFB2 (transforming growth factor, beta 2); IL6 and KRTl8 (keratin l8); IER3 (immediate early response 3), MEST (mesoderm speciﬁc transcript homolog) and TGFB2; CD200 and IER3; CD200 and IL6; CD200 and KRTl8; CD200 and LRAP; CD200 and MEST; CD200 and NFE2L3 (nuclear factor (erythroid-derived 2)-like 3); or CD200 and TGFB2 at a detectably higher level than an equivalent number of bone marrow- derived mesenchymal stem cells wherein said bone marrow-derived mesenchymal stem cells have one a number of passages in culture equivalent to the number of passages said isolated placental stem cells have undergone. In other speciﬁc embodiments, the arPSCs express ARTS-l, CD200, IL6 and LRAP; ARTS-l, IL6, TGFB2, IER3, KRTl8 and MEST; CD200, IER3, IL6, KRTl8, LRAP, MEST, NFE2L3, and TGFB2; ARTS-l, CD200, IER3, IL6, KRTl8, LRAP, MEST, NFE2L3, and TGFB2; or IER3, MEST and TGFB2 at a detectably higher level than an equivalent number of bone marrow-derived mesenchymal stem cells, wherein said bone -derived hymal stem cells have undergone a number of passages in culture equivalent to the number of passages said isolated arPSCs have undergone. 2013/074892 Expression of the above-referenced genes can be assessed by standard techniques.

For example, probes based on the ce of the gene(s) can be individually selected and constructed by conventional techniques. Expression of the genes can be assessed, e.g., on a microarray comprising probes to one or more of the genes, e.g., an Affymetrix GENECHIP® Human Genome Ul33A 2.0 array, or an Affymetrix GENECHIP® Human Genome U133 Plus 2.0 (Santa Clara, California). sion of these genes can be assessed even if the sequence for a ular GenBank accession number is amended because probes speciﬁc for the amended sequence can readily be ted using well-known standard techniques.

The level of expression of these genes can be used to conﬁrm the identity of a population of isolated arPSCs, to identify a population of cells as comprising at least a plurality of isolated arPSCs, or the like. Populations of isolated , the identity of which is conﬁrmed, can be clonal, e.g., populations of isolated arPSCs ed from a single isolated arPSC, or a mixed population of arPSCs, e.g., a population of cells comprising isolated arPSCs that are expanded from multiple isolated arPSCs, or a population of cells comprising isolated arPSCs, as described herein, and at least one other type of cell.

The level of expression of these genes can be used to select populations of isolated arPSCs. For example, a population of cells, e.g., clonally-expanded arPSCs, may be selected if the expression of one or more of the genes listed above is signiﬁcantly higher in a sample from the population of cells than in an lent population of bone marrow-derived mesenchymal stem cells. Such selecting can be of a population from a plurality of isolated arPSC populations, from a plurality of cell populations, the identity of which is not known, etc.

Isolated arPSCs can be selected on the basis of the level of expression of one or more such genes as ed to the level of expression in said one or more genes in, e.g., a bone marrow-derived mesenchymal stem cell control. In one embodiment, the level of expression of said one or more genes in a sample comprising an equivalent number of bone marrow-derived mesenchymal stem cells is used as a control. In another ment, the control, for isolated arPSCs tested under certain conditions, is a numeric value representing the level of expression of said one or more genes in bone marrow-derived mesenchymal stem cells under said ions.

Similarly, the expression of anoikis ated genes can be used to select populations of isolated arPSCs. For example, a population of cells, e.g., ly-expanded arPSCs, may be selected if the expression of one or more anoikis associated genes (e.g., one or rmmoﬁmmmmmwwmmdgmmwmﬂmmmwﬁﬁwmwmﬁnmmmbﬁmnm population of cells relative an equivalent population of unmodiﬁed placental stem cells.

The isolated arPSCs described herein display the above characteristics (e.g., combinations of cell surface markers and/or gene expression s) in y culture, or during eration in medium comprising, e.g., DMEM-LG (Gibco), 2% fetal calf serum (FCS) (Hyclone Laboratories), lx insulin-transferrin-selenium (ITS), lx ic-acid-bovine-serum- albumin (LA-BSA), 10'9 M dexamethasone (Sigma), 10'4M ascorbic acid 2-phosphate (Sigma), epidermal growth factor (EGF) lOng/ml (R&D Systems), platelet derived-growth factor (PDGFBB ) lOng/ml (R&D Systems), and 100U penicillin/ l OOOU streptomycin.

In certain embodiments of any of the arPSCs disclosed herein, the cells are human. In certain embodiments of any of the arPSCs disclosed herein, the cellular marker characteristics or gene expression characteristics are human markers or human genes.

In another specific embodiment of the isolated arPSCs or populations of cells comprising the isolated arPSCs, said cells or population have been expanded, for example, passagedatleay,abouu,ornornorethan,l,2,3,4,5,6,7,8,9,lO,ll,lZ,l3,l4,15,l6,l7,18, l9,or20tnnes,orprohfennedforatleam;abouu,ornoinorethan,l,2,3,4,5,6,7,8,9,10,12, l4,l6,18,20,22,24,26,28,30,32,34,36,38culﬂ)popukunn1doubhngs.Inznunherspecﬂic embodiment of said ed arPSCs or populations of cells comprising the isolated arPSCs, said cells or population are y isolates. In another specific embodiment of the isolated arPSCs, or populations of cells comprising isolated arPSCs, that are disclosed herein, said isolated arPSCs are fetal in origin (that is, have the fetal pe).

In certain embodiments, said ed arPSCs do not differentiate during culturing in growth medium, z'.e., medium formulated to promote proliferation, e.g., during proliferation in growth . In another specific embodiment, said isolated arPSCs do not require a feeder layer in order to proliferate. In another ic embodiment, said isolated arPSCs do not differentiate in culture in the absence of a feeder layer, solely because of the lack of a feeder cell layer.

In another embodiment, the ed arPSCs are positive for aldehyde dehydrogenase (ALDH), as assessed by an aldehyde dehydrogenase ty assay. Such assays are known in the art (see, e.g., Bostian and Betts, m. J., 173, 787, (1978)). In a speciﬁc embodiment, said ALDH assay uses ALDEFLUOR® (Aldagen, Inc., Ashland, Oregon) as a marker of aldehyde dehydrogenase activity. In a speciﬁc embodiment, between about 3% and about 25% of arPSCs are positive for ALDH. In another embodiment, said isolated arPSCs show at least three-fold, or at least ﬁve-fold, higher ALDH activity than a population of bone marrow-derived mesenchymal stem cells having about the same number of cells and cultured under the same conditions.

In n embodiments of any of the populations of cells comprising the isolated arPSCs described herein, the arPSCs in said populations of cells are substantially ﬁee of cells having a maternal genotype; e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the arPSCs in said population have a fetal genotype. In certain other embodiments of any of the populations of cells comprising the ed arPSCs described herein, the populations of cells comprising said arPSCs are substantially free of cells having a al genotype; e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the cells in said population have a fetal genotype.

In a speciﬁc embodiment of any of the above isolated arPSCs or cell tions comprising ed arPSCs, the karyotype of the cells, e.g., all of the cells, or at least about 95% or about 99% of the cells in said population, is normal. In another c embodiment of any of the above arPSCs or populations or arPSCs, the arPSCs are non-matemal in .

In a speciﬁc embodiment of any of the embodiments of placental cells disclosed , the placental cells are genetically stable, displaying a normal diploid chromosome count and a normal karyotype.

Isolated arPSCs, or populations of isolated arPSCs, bearing any of the above combinations of s, can be combined in any ratio. Any two or more of the above isolated arPSCs populations can be combined to form an isolated arPSC population. For example, a population of isolated arPSCs can comprise a ﬁrst population of isolated arPSCs deﬁned by one of the marker combinations bed above, and a second population of isolated arPSCs deﬁned by another of the marker combinations described above, wherein said ﬁrst and second populations are combined in a ratio of about 1:99, 2:98, 3:97, 4:96, 5:95, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, or about 99:l. In like fashion, any three, four, ﬁve or more of the above-described isolated arPSCs or isolated placental stem cell populations can be combined.

Isolated placental stem cells useful in methods for generating the arPSCs described herein can be obtained, e.g., by disruption of placental tissue, with or without enzymatic digestion or perﬁJsion. For example, populations of isolated placental stem cells can be produced according to a method comprising perfusing a mammalian placenta that has been drained of cord blood and perfused to remove residual blood; perfusing said ta with a perfusion solution; and collecting said ion on, wherein said perfusion solution after perfusion comprises a population of placental cells that comprises isolated placental stem cells; and isolating said placental stem cells from said population of cells. In a ic embodiment, the perﬁlsion solution is passed through both the umbilical vein and umbilical arteries and collected after it exudes from the placenta. In another specific embodiment, the perfusion solution is passed through the umbilical vein and collected from the umbilical arteries, or passed through the umbilical arteries and collected from the umbilical vein.

In various embodiments, the isolated placental stem cells, useful in methods for generating the arPSCs described herein contained within a population of cells obtained from perfusion of a placenta, are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% of said population of placental stem cells. In another specific ment, the isolated placental stem cells ted by perfusion comprise fetal and maternal cells. In another specific embodiment, the isolated placental stem cells collected by perfusion are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% fetal cells.

In another specific ment, provided herein is a composition comprising a tion of the isolated placental stem cells useful in methods for generating the arPSCs described herein, collected (isolated) by perﬁJsion, wherein said composition comprises at least a portion of the perﬁJsion solution used to isolate the placental stem cells.

Populations of the ed placental stem cells useful in s for generating the arPSCs described herein can be produced by digesting placental tissue with a tissue- disrupting enzyme to obtain a population of placental cells comprising the placental stem cells, and isolating, or substantially isolating, a plurality of the placental stem cells from the remainder of said placental cells. The whole, or any part of, the placenta can be digested to obtain the isolated placental stem cells described herein. In specific ments, for example, said tal tissue can be a whole ta (e.g., including an umbilical cord), an amniotic membrane, chorion, a combination of amnion and chorion, or a combination of any of the WO 93753 2013/074892 foregoing. In other speciﬁc embodiments, the tissue-disrupting enzyme is trypsin or collagenase.

In various ments, the isolated placental stem cells, contained Within a population of cells ed from digesting a placenta, are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% of said population of placental cells.

The populations of isolated arPSCs described above, and populations of isolated arPSCs lly, can comprise about, at least, or no more than, 1 x 105, 5 x 105, l x 106, 5 x 106, 1 x1015 x107,1x108,5 x108,1x109,5 x109,1x101°,5 x 1010, 1 x 1011 or more ofthe isolated arPSCs. Populations of isolated arPSCs useful in the methods and compositions described herein comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% viable isolated placental stem cells, e.g., as determined by, e.g., trypan blue exclusion.

For any of the above tal stem cells, or populations of placental stem cells, (e.g., unmodiﬁed placental stem cells useful in methods of ing the arPSCs described herein, or the arPSCs described , or compositions thereof) the cells or population of placental stem cells are, or can se, cells that have been passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, l2, l4, l6, 18, or 20 times, or more, or expanded for l, 2, 3, 4, 5, 6, 7, 8, 9, 10, l2, l4, l6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 population doublings, or more.

In a speciﬁc embodiment of any of the above placental stem cells or placental stem cells populations (e.g., unmodiﬁed placental stem cells useful in methods of producing the arPSCs described herein, or the arPSCs described herein, or compositions thereof), the karyotype of the cells, or at least about 95% or about 99% of the cells in said population, is normal. In another speciﬁc embodiment of any of the above placental stem cells or placental stem cells populations (e.g., ﬁed placental stem cells useful in methods of producing the arPSCs bed herein, or the arPSCs bed herein, or compositions thereof), the cells, or cells in the population of cells, are non-matemal in origin.

Isolated placental stem cells, or populations of isolated placental stem cells, (e. g., unmodiﬁed placental stem cells useful in methods of producing the arPSCs described , or the arPSCs described herein, or compositions thereof) bearing any of the above combinations of markers, can be combined in any ratio. Any two or more of the above placental stem cells populations can be isolated, or enriched, to form a placental stem cells population. For example, an population of isolated placental stem cells comprising a ﬁrst population of placental stem cells WO 93753 deﬁned by one of the marker combinations described above can be combined with a second population of placental stem cells deﬁned by another of the marker combinations described above, wherein said ﬁrst and second populations are combined in a ratio of about 1:99, 2:98, 3:97, 4:96, 5:95, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, or about 99:1. In like n, any three, four, ﬁve or more of the described tal stem cells or placental stem cells populations can be combined.

In a speciﬁc embodiment of the above-mentioned placental stem cells (e. g., ﬁed placental stem cells useful in methods of producing the arPSCs described , or the arPSCs described herein, or compositions thereof), the placental stem cells constitutively secrete IL-6, IL-8 and monocyte chemoattractant n (MCP-l).

The suppressive pluralities of arPSCs described above can comprise about, at least, or no more than, 1 x 105, 5 x105,1x106,5 x106,1x107,5 x107,1x108,5 x 108,1x109,5 x109,1x1010,5 x 1010, 1 x 1011 or more arPSCs.

In certain embodiments, the arPSCs useful in the methods provided herein, do not express CD34, as detected by immunolocalization, after exposure to 1 to 100 ng/mL VEGF for 4 to 21 days. In another speciﬁc ment, said arPSCs induce endothelial cells to form sprouts or tube-like structures, e.g., when cultured in the presence of an angiogenic factor such as vascular elial growth factor (VEGF), epithelial growth factor (EGF), platelet derived growth factor (PDGF) or basic ﬁbroblast growth factor (bFGF), e. g., on a substrate such as MATRIGELTM.

In another aspect, the arPSCs provided herein, or a population of cells, e.g., a population of arPSCs, or a population of cells wherein at least about 50%, 60%, 70%, 80%, 90%, 95% or 98% of cells in said tion of cells are arPSCs, secrete one or more, or all, ofVEGF, HGF, IL-8, MCP-3, FGF2, follistatin, G-CSF, EGF, ENA-78, GRO, IL-6, MCP-l, PDGF-BB, TIMP-2, uPAR, or galectin-l, e.g., into culture medium in which the cell, or cells, are grown. In another embodiment, the arPSCs express increased levels of CD202b, IL-8 and/or VEGF under hypoxic conditions (e.g., less than about 5% 02) compared to normoxic conditions (e.g., about % or about 21% 02).

In another embodiment, any of the arPSCs or populations of cells sing arPSCs described herein can cause the formation of sprouts or tube-like structures in a population of endothelial cells in contact with said arPSCs. In a speciﬁc embodiment, the WO 93753 arPSCs are co-cultured with human endothelial cells, which form sprouts or tube-like structures, or support the formation of endothelial cell s, e.g., when cultured in the presence of extracellular matrix proteins such as collagen type I and IV, and/or angiogenic factors such as vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), platelet derived growth factor (PDGF) or basic fibroblast growth factor (bFGF), e. g., in or on a substrate such as placental collagen or MATRIGELTM for at least 4 days. In another embodiment, any of the populations of cells comprising arPSCs described herein secrete angiogenic factors such as vascular elial growth factor , hepatocyte growth factor (HGF), platelet derived growth factor , basic fibroblast growth factor (bFGF), or Interleukin-8 (IL-8) and thereby can induce human endothelial cells to form sprouts or tube-like structures when cultured in the ce of extracellular matrix proteins such as collagen type I and IV e.g., in or on a substrate such as placental collagen or MATRIGELTM.

In another embodiment, any of the above populations of cells comprising arPSCs secretes angiogenic factors. In specific embodiments, the population of cells secretes vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and/or interleukin-8 (IL-8). In other specific embodiments, the population of cells comprising arPSCs secretes one or more angiogenic factors and thereby induces human endothelial cells to migrate in an in vitro wound healing assay. In other specific embodiments, the population of cells comprising arPSCs induces tion, differentiation or proliferation of human elial cells, endothelial itors, myocytes or sts.

In another embodiment, provided herein are arPSCs, and populations of arPSCs, wherein said arPSCs comprise any of the foregoing characteristics (e.g., are CD347, CDlO+, CD105+ and CDZOOI), and wherein at least one anoikis associated gene is downregulated/inhibited in said arPSCs relative to the level of expression of said anoikis associated gene in an lent number of unmodified placental stem cells (e. g., CD347, CDlOI, CD105+ and CD200+ unmodified placental stem cells). In a specific embodiment, the at least one anoikis associated gene is AMIGOl (NCBI GENE ID NO:57463); 20 (NCBI GENE ID 69); CD38 (NCBI GENE ID ); CLCCl (NCBI GENE ID NO:23155); CNTF (NCBI GENE ID NO: 1270); ZFP9l-CNTF (NCBI GENE ID NO:386607); COX8A (NCBI GENE ID NO: 135 l); DHX34 (NCBI GENE ID NO:9704); FAMl75A (NCBI GENE ID NO:NO 51023); MRPS18C (NCBI GENE ID NO:84142); FAM44C (NCBI GENE ID NO:284257); FBP2 (NCBI GENE ID NO:8789); FLIl (NCBI GENE ID NO:2313); FREM3 (NCBI GENE ID NO: 166752); IFIT5 (NCBI GENE ID NO:24138); LOC399851 (NCBI GENE ID NO:399851); LOC400713 (NCBI GENE ID NO:400713); LOC651610 (NCBI GENE ID NO:651610); PIGP (NCBI GENE ID NO:51227); SH3TC2 (NCBI GENE ID NO:79628); SLC2A3 (NCBI GENE ID 5); STAU2 (NCBI GENE ID NO:27067) TMEFF1 (NCBI GENE ID 7); TMEM217 (NCBI GENE ID NO:221468); TMEM79 (NCBI GENE ID NO:84283); USHBP1 (NCBI GENE ID NO:83878);APH1B (NCBI GENE ID NO:83464); ATP2B2 (NCBI GENE ID ); C13orf39 (NCBI GENE ID NO: 196541); C4orf17 (NCBI GENE ID NOi84103); C4orf46 (NCBI GENE ID NO:201725); DDX41 (NCBI GENE ID NO:51428); DKFZp547J222 (NCBI GENE ID NO:84237); FGFR1 (NCBI GENE ID NO:2260); FHDC1 (NCBI GENE ID NO:85462); GNAI2 (NCBI GENE ID NO:2771); GP5 (NCBI GENE ID NO:2814); ILlRN (NCBI GENE ID NO:3557); KIF24 (NCBI GENE ID NO:347240); KNDC1 (NCBI GENE ID NO:85442); LOC100132598 (NCBI GENE ID NO: 100132598); LOC151760 (NCBI GENE ID NO:151760); LOC152024 (NCBI GENE ID NO: 152024); 833 (NCBI GENE ID NO:339833); LPAR4 (NCBI GENE ID NO:2846); LSG1 (NCBI GENE ID NOi5534l); MAP3K5 (NCBI GENE ID NO:4217); PDK3 (NCBI GENE ID NO:5165); PELI2 (NCBI GENE ID NO:57161); RNF103 (NCBI GENE ID NO:7844); SNX31 (NCBI GENE ID NO: 169166); TXN2 (NCBI GENE ID NO:25828); or XKR7 (NCBI GENE ID 702). In a speciﬁc embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of said anoikis associated genes are gulated/inhibited in said arPSCs relative to the level of expression of said anoikis ated gene(s) in an equivalent number of unmodiﬁed placental stem cells (e. g., CD343 CD101 CD105+ and CD200+ unmodiﬁed placental stem .

In another speciﬁc embodiment, provided herein is an isolated CD347, CD10+, CD105+ and CD200+ arPSC, wherein said arPSC expresses the anoikis associated gene FHDC1 (NCBI GENE ID NO:85462) at a sed level as compared to the expression of the s associated gene FHDC1 (NCBI GENE ID NO:85462) in an ﬁed placental stem cell. In another speciﬁc embodiment, provided herein is an isolated CD347, CD10+, CD105+ and CD200+ arPSC, wherein said arPSC expresses the anoikis associated gene GNAI2 (NCBI GENE ID NO:2771) at a decreased level as compared to the expression of the anoikis associated gene GNAI2 (NCBI GENE ID NO:2771) in an unmodiﬁed placental stem cell. In another speciﬁc embodiment, provided herein is an isolated CD34’, CD10: CD105+ and CD200+ arPSC, wherein said arPSC expresses the anoikis associated gene KNDCl (NCBI GENE ID 42) at a decreased level as compared to the expression of the anoikis associated gene KNDCl (NCBI GENE ID NO:85442) in an unmodiﬁed placental stem cell. In another speciﬁc ment, provided herein is an isolated CD34’, CD10: CD105+ and CD200+ arPSC, wherein said arPSC expresses the anoikis associated gene LPAR4 (NCBI GENE ID NO:2846) at a decreased level as compared to the expression of the anoikis associated gene LPAR4 (NCBI GENE ID NO:2846) in an unmodiﬁed placental stem cell. In another speciﬁc embodiment, provided herein is an isolated CD34’, CD10: CD105+ and CD200+ arPSC, wherein said arPSC ses the anoikis associated gene MAP3K5 (NCBI GENE ID NO:4217) at a sed level as ed to the expression of the anoikis associated gene MAP3K5 (NCBI GENE ID NO:4217) in an unmodiﬁed placental stem cell. In another speciﬁc embodiment, ed herein is an isolated CD34’, CD10: CD105+ and CD200+ arPSC, wherein said arPSC ses the anoikis associated gene SLC2A3 (NCBI GENE ID NO:6515) at a decreased level as compared to the expression of the anoikis associated gene SLC2A3 (NCBI GENE ID NO:6515) in an unmodiﬁed placental stem cell. In another speciﬁc ment, provided herein is an ed CD347, CD10+, CD105+ and CD200+ arPSC, wherein said arPSC expresses the anoikis associated gene STAUZ (NCBI GENE ID NO:27067) at a decreased level as compared to the expression of the anoikis associated gene STAU2 (NCBI GENE ID NO:27067) in an unmodiﬁed placental stem cell. Further provided herein are populations of cells comprising such arPSCs and compositions comprising such arPSCs.

In another speciﬁc embodiment, provided herein is an isolated CD347, CD10+, CD105+ and CD200+ arPSC, wherein said arPSC ses one, two, three, or more of the following placental stem cell s-associated genes at a decreased level as compared to the expression of the same anoikis associated gene(s) in an unmodiﬁed placental stem cell: FHDCl (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID NO:2771), KNDCl (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID 7), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID 67). In another speciﬁc embodiment, provided herein is an isolated CD34’, CD10: CD105+ and CD200+ arPSC, wherein said arPSC (i) expresses one, two, three, or more of the following placental stem cell anoikis-associated genes at a decreased level as compared to the expression of the same anoikis associated gene(s) in an unmodiﬁed placental stem cell: FHDCl (NCBI GENE ID NO:85462), GNA12 (NCBI GENE ID NO:277l), KNDCl (NCBI GENE ID 42), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), and STAU2 (NCBI GENE ID NO:27067); and (ii) expresses at least one additional anoikis associated gene d in Table l at a sed level as compared to the expression of the same anoikis associated gene(s) in an unmodiﬁed placental stem cell. Further provided herein are populations of cells comprising such arPSCs and compositions comprising such arPSCs. .3.3 Growth in Culture The growth of the placental cells, including the arPSCs bed , as for any mammalian cell, s in part upon the particular medium selected for growth. During culture, the placental stem cells used in the methods of production of the arPSCs provided herein adhere to a substrate in culture, e.g. the surface of a tissue culture container (e.g., tissue culture dish plastic, ctin-coated plastic, and the like) and form a monolayer. In the absence of a substrate for the placental stem cells to adhere to (e.g., under tachment conditions), the placental stem cells undergo anoikis, and demonstrate diminished survival. In contrast, the arPSCs described herein do not undergo anoikis in the absence of a substrate for the arPSCs to adhere to (e.g., under low-attachment conditions), and thus demonstrate sed survival in such conditions relative to unmodiﬁed placental stem cells.

In a speciﬁc embodiment, the arPSCs described herein demonstrate sed survival ve to ﬁed placental stem cells when cultured under low attachment conditions in vitro, e. g., when cultured in low-attachment tissue culture . In another speciﬁc ment, the arPSCs described herein demonstrate increased survival relative to unmodiﬁed placental stem cells when cultured under low attachment conditions in vivo, e.g., When administered to a subject systemically or locally, or by another administration method wherein the cells are administered in a low attachment environment.

In certain embodiments, when cultured under low-attachment conditions (either in vitro or in vivo), the arPSCs described herein demonstrate at least a 1.5-fold, 2-fold, 25-fold, 3- fold, 35-fold, 4-fold, 45-fold, S-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increase in survival relative to an equivalent amount of unmodiﬁed placental stem cells cultured under the same conditions. In certain embodiments, when cultured under low-attachment conditions (either in vitro or in vivo), the arPSCs described herein demonstrate a l.5-fold to ld, a 2- fold to 3-fold, a 25-fold to 35-fold, a 3-fold to 4-fold, a 35-fold to 4.5-fold, a 4-fold to 5-fold, a -fold to 6-fold, a 6-fold to 7-fold, a 7-fold to 8-fold, an 8-fold to 9-fold, or a 9-fold to 10-fold increase in al relative to an equivalent amount of unmodiﬁed placental stem cells ed under the same conditions. In another speciﬁc embodiment, when cultured under low- attachment conditions (either in vitro or in vivo), the arPSCs described herein demonstrate a greater than 10-fold increase in survival ve to an equivalent amount of unmodiﬁed placental stem cells ed under the same conditions. Survival of the arPSCs and unmodiﬁed placental stem cells can be assessed using methods known in the art, e.g., trypan blue exclusion assay, ﬁuorescein diacetate uptake assay, propidium iodide uptake assay; thymidine uptake assay, and MTT (3-(4,5-Dimethylthiazolyl)—2,5-diphenyltetrazolium bromide) assay. .4 METHODS OF OBTAINING PLACENTAL STEM CELLS FOR USE IN METHODS OF GENERATING ANOIKIS-RESISTANT PLACENTAL STEM CELLS .4.1 Stem Cell Collection Composition Placental stem cells for use in the methods of generating arPSCs described herein can be collected and ed according to the methods provided herein. Generally, placental stem cells are obtained from a mammalian placenta using a physiologically-acceptable solution, e. g., a stem cell tion ition. A stem cell collection composition is bed in detail in related US. Patent Application Publication No. 20070190042.

The stem cell tion composition can comprise any physiologically-acceptable 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, s solution, 0.9% NaCl. etc), a culture medium (e.g., DMEM, HDMEM, etc), and the like.

The stem cell collection composition can comprise one or more components that tend to preserve placental stem cells, that is, t the placental stem cells from dying, or delay the death of the placental stem cells, reduce the number of placental stem cells in a population of cells that die, or the like, from the time of collection to the time of culturing. Such ents can be, e.g., an apoptosis inhibitor (e.g., a caspase inhibitor or INK inhibitor); a vasodilator (e.g., magnesium sulfate, an antihypertensive drug, atrial natriuretic peptide (ANP), adrenocorticotropin, corticotropin-releasing hormone, sodium russide, azine, adenosine sphate, adenosine, thacin or magnesium sulfate, a phosphodiesterase inhibitor, etc); a necrosis inhibitor (e.g., 2-(lH-Indolyl)pentylamino-maleimide, pyrrolidine carbamate, or clonazepam); a TNF-(x inhibitor; and/or an oxygen-carrying perﬂuorocarbon (e.g., perﬂuorooctyl bromide, rodecyl bromide, etc).

The stem cell collection composition can comprise one or more tissue-degrading enzymes, e.g., a oprotease, a serine protease, a neutral protease, an RNase, or a DNase, or the like. Such enzymes include, but are not limited to, enases (e.g., enase I, II, III or IV, a collagenase from Clostridium histolytz'cum, etc); dispase, thermolysin, se, trypsin, LIBERASE, hyaluronidase, and the like.

The stem cell collection composition can comprise a bacteriocidally or bacteriostatically 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, cefprozil, or, cefixime or cefadroxil), a clarithromycin, an erythromycin, a llin (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 also comprise one or more of the following compounds: ine (about 1 mM to about 50 mM); D-glucose (about 20 mM to about 100 mM); magnesium ions (about 1 mM to about 50 mM); a macromolecule of molecular weight greater than 20,000 daltons, in one embodiment, present in an amount sufficient to maintain elial integrity and cellular Viability (e.g., a synthetic or naturally occurring colloid, a polysaccharide such as dextran or a hylene glycol present at about 25 g/l to about 100 g/l, or about 40 g/l to about 60 g/l); an antioxidant (e.g., butylated hydroxyanisole, butylated hydroxytoluene, glutathione, Vitamin C or Vitamin E present at about 25 uM to about 100 uM); a reducing agent (e.g., N—acetylcysteine present at about 0.1 mM to about 5 mM); an agent that prevents calcium entry into cells (e.g., verapamil present at about 2 uM to about 25 uM); nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L); an anticoagulant, in one embodiment, present in an amount sufﬁcient to help prevent clotting of residual blood (6.g. , heparin or hirudin present at a concentration of about 1000 units/l to about 100,000 units/l); or an amiloride containing compound (e.g., amiloride, ethyl isopropyl amiloride, hexamethylene amiloride, dimethyl amiloride or isobutyl amiloride present at about 1.0 uM to about 5 uM). 52WM Generally, a human placenta is recovered y after its expulsion after birth. In a preferred embodiment, the placenta is recovered from a patient after ed consent and after a complete medical history of the patient is taken and is associated with the placenta. Preferably, the medical y continues after delivery. Such a medical history can be used to coordinate subsequent use of the placenta or the stem cells harvested therefrom. For e, human placental cells can be used, in light of the medical history, for personalized medicine for the infant associated with the placenta, or for parents, siblings or other relatives of the infant.

Prior to ry of placental stem cells, the umbilical cord blood and placental blood are removed. In certain ments, after delivery, the cord blood in the ta is recovered. The placenta can be ted to a conventional cord blood recovery s.

Typically a needle or cannula is used, with the aid of y, to uinate the placenta (see, e.g., Anderson, US. Patent No. 5,372,581; Hessel et al., US. Patent No. 5,415,665). The needle or cannula is usually placed in the umbilical vein and the placenta can be gently massaged to aid in draining cord blood from the placenta. Such cord blood recovery may be performed commercially, e.g, nk Inc., Cedar Knolls, N.J., ViaCord, Cord Blood Registry and Cryocell. Preferably, the placenta is gravity drained without further manipulation so as to minimize tissue disruption during cord blood recovery.

Typically, a placenta is transported from the delivery or birthing room to another location, e.g., a laboratory, for recovery of cord blood and collection of stem cells by, e.g., perfusion or tissue dissociation. The placenta is preferably transported in a sterile, thermally insulated transport device (maintaining the temperature of the placenta between 20-280C), for example, by placing the placenta, with clamped proximal umbilical cord, in a sterile zip-lock plastic bag, which is then placed in an insulated container. In another embodiment, the placenta is transported in a cord blood collection kit substantially as bed in pending United States patent application no. 11/230,760, filed September 19, 2005. Preferably, the placenta is delivered to the laboratory four to twenty-four hours ing delivery. In certain embodiments, the proximal umbilical cord is clamped, ably within 4-5 cm (centimeter) of the insertion into the placental disc prior to cord blood recovery. In other embodiments, the proximal umbilical cord is clamped after cord blood recovery but prior to r processing of the placenta.

The placenta, prior to placental stem cell collection, can be stored under sterile conditions and at either room temperature or at a temperature of 5 to 25°C (centigrade). The placenta may be stored for a period of longer than forty eight hours, and preferably for a period of four to -four hours prior to perﬁasing the ta to remove any residual cord blood.

The placenta is preferably stored in an anticoagulant solution at a temperature of 5 to 25°C grade). Suitable anticoagulant solutions are well known in the art. For example, a solution of n or in sodium can be used. In a preferred embodiment, the anticoagulant solution comprises a solution of heparin (e.g., 1% w/w in 1:1000 solution). The exsanguinated placenta is preferably stored for no more than 36 hours before placental cells are collected.

The mammalian placenta or a part thereof, once collected and prepared generally as above, can be treated in any art-known manner, e.g., can be perfused or disrupted, e.g., digested with one or more tissue-disrupting enzymes, to obtain stem cells. .4.3 Physical Disruption and tic Digestion of Placental Tissue In one ment, placental stem cells are collected from a mammalian ta by physical disruption, e.g., enzymatic digestion, of the organ, e.g., using the stem cell collection composition described above. For example, the ta, or a portion thereof, may be, e.g., crushed, sheared, minced, diced, chopped, macerated or the like, while in contact with, e.g., a buffer, medium or a stem cell collection composition, and the tissue subsequently digested with one or more enzymes. The placenta, or a portion thereof, may also be physically ted and digested with one or more enzymes, and the resulting material then immersed in, or mixed into, a buffer, medium or a stem cell collection composition. Any method of physical disruption can be used, provided that the method of disruption leaves a plurality, more preferably a majority, and more preferably at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in said organ viable, as determined by, e.g., trypan blue ion.

Typically, placental cells can be obtained by disruption of a small block of placental , e.g., a block ofplacental tissue that is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or about 1000 cubic millimeters in volume.

Enzymatic digestion can be performed using single enzymes or combinations of enzymes. In one embodiment, enzymatic digestion of placental tissue uses a combination of a matrix metalloprotease, a neutral protease, and a mucolytic enzyme for ion of hyaluronic acid, such as a combination of collagenase, dispase, and hyaluronidase or a combination of LIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.) and hyaluronidase. Other enzymes that can be used to t placenta tissue include papain, ibonucleases, serine proteases, such as trypsin, rypsin, or elastase. Serine proteases may be inhibited by alpha 2 microglobulin in serum and therefore the medium used for digestion is usually serum-free.

EDTA and DNase are commonly used in enzyme ion procedures to increase the efficiency of cell recovery. The digestate is preferably diluted so as to avoid trapping stem cells within the viscous digest.

Typical concentrations for tissue digestion enzymes include, e.g., 50-200 U/mL for collagenase I and collagenase IV, 1-10 U/mL for dispase, and 10-100 U/mL for elastase.

Proteases can be used in combination, that is, two or more proteases in the same digestion reaction, or can be used sequentially in order to liberate placental cells. For example, in one embodiment, a placenta, or part thereof, is digested first with an appropriate amount of collagenase I at 2 mg/ml for 30 minutes, followed by digestion with trypsin, 0.25%, for 10 minutes, at 37°C. Serine proteases are preferably used consecutively following use of other In r ment, the tissue can ﬁarther be disrupted by the addition of a chelator, e.g., ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA) to the stem cell collection composition comprising the stem cells, or to a solution in which the tissue is disrupted and/or ed prior to isolation of the placental stem cells with the stem cell tion composition.

It will be appreciated that where an entire placenta, or portion of a placenta comprising both fetal and maternal cells (for example, where the n of the placenta comprises the chorion or cotyledons) is digested to obtain placental stem cells, the placental cells collected will comprise a mix of placental cells derived from both fetal and al s.

Where a portion of the placenta that comprises no, or a negligible number of, maternal cells (for example, amnion) is used to obtain placental stem cells, the placental stem cells collected will comprise almost exclusively fetal tal stem cells. .4.4 Placental Perfusion Placental stem cells can also be obtained by perﬁasion of the mammalian placenta.

Methods of perﬁlsing mammalian placenta to obtain stem cells are disclosed, e.g., in , US.

Application Publication No. 2002/0123141, and in related US. Provisional Application No. 60/754,969, ed “Improved Composition for Collecting and Preserving Placental Cells and Methods of Using the Composition” ﬁled on December 29, 2005. tal stem cells can be collected by perfusion, e.g., through the placental vasculature, using, e.g., a stem cell collection composition as a perfusion solution. In one embodiment, a mammalian placenta is perfused by passage of ion solution through either or both of the umbilical artery and umbilical vein. The ﬂow of perﬁasion solution through the placenta may be accomplished using, e.g., gravity ﬂow into the placenta. Preferably, the perfusion solution is forced h the placenta using a pump, e.g., a altic pump. The umbilical vein can be, e.g., cannulated with a a, e.g., a TEFLON® or plastic cannula, that is connected to a sterile connection apparatus, such as sterile tubing. The sterile tion tus is connected to a perfusion manifold.

In preparation for ion, the placenta is preferably oriented (e.g., suspended) in such a manner that the umbilical artery and umbilical vein are located at the highest point of the placenta. The placenta can be perfused by passage of a perfusion ﬂuid, e.g., the stem cell collection composition ed herein, through the placental vasculature, or through the placental vasculature and surrounding tissue. In one embodiment, the umbilical artery and the umbilical vein are connected simultaneously to a pipette that is connected via a ﬂexible connector to a reservoir of the perfusion solution. The perfusion solution is passed into the umbilical vein and artery. The perfusion solution exudes from and/or passes through the walls of the blood s into the nding tissues of the ta, and is collected in a suitable open vessel from the surface of the placenta that was attached to the uterus of the mother during gestation. The perfusion solution may also be introduced through the umbilical cord opening and allowed to flow or percolate out of openings in the wall of the placenta which interfaced with the al uterine wall. In another embodiment, the perfusion solution is passed through 2013/074892 the umbilical veins and ted from the umbilical artery, or is passed through the umbilical artery and collected from the umbilical veins.

In one embodiment, the proximal umbilical cord is clamped during perfusion, and more ably, is clamped within 4-5 cm (centimeter) of the cord’s insertion into the placental disc.

The first collection of perfusion ﬂuid from a mammalian placenta during the exsanguination process is generally colored with residual red blood cells of the cord blood and/or placental blood; this portion of the perfusion can be discarded. The ion ﬂuid becomes more colorless as perfusion proceeds and the residual cord blood cells are washed out of the placenta.

The volume of perfusion liquid used to collect placental stem cells may vary ing upon the number of placental stem cells to be collected, the size of the placenta, the number of collections to be made from a single placenta, etc. In various ments, the volume of perfusion liquid may be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 InL,1001nLto20001nL,250nﬂgu)20001nL,5001nLto20001nL,or7501nLto2000nﬂ; Typically, the placenta is perfused with 700-800 mL of perfusion liquid following exsanguination.

The placenta can be perfused a plurality of times over the course of several hours or several days. Where the placenta is to be perfused a plurality of times, it may be ined or ed under aseptic conditions in a container or other suitable vessel, and perfused with the stem cell collection composition, or a standard perfusion solution (e.g., a normal saline solution such as ate buffered saline (“PBS”)) with or without an agulant (e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin), and/or with or without an antimicrobial agent (e.g., B-mercaptoethanol (0.1 mM); antibiotics such as streptomycin (e.g., at 40-100 ug/ml), penicillin (e.g., at 40U/ml), amphotericin B (e.g., at 0.5 ug/ml). In one embodiment, an isolated ta is ined or cultured for a period of time without collecting the perﬁJsate, such that the placenta is maintained or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3 or more days before perfusion and collection of perfusate. The perfused placenta can be maintained for one or more additional time(s), e.g., 1, 2, 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24(n1nonzhoungand perfused a second time with, e.g., 700-800 mL perfusion ﬂuid. The placenta can be perfused 1, 2013/074892 2, 3, 4, 5 or more times, for example, once every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment, perfusion of the placenta and tion of perfusion solution, e.g., stem cell collection composition, is repeated until the number of recovered nucleated cells falls below 100 cells/ml. The perfusates at different time points can be ﬁthher processed individually to recover time-dependent populations of placental stem cells. Perfusates from different time points can also be .

Without wishing to be bound by any theory, after exsanguination and a sufﬁcient time of perfusion of the placenta, placental stem cells are believed to migrate into the exsanguinated and ed microcirculation of the placenta where they are collectable, preferably by washing into a collecting vessel by perfusion. Perﬁlsing the isolated placenta not only serves to remove al cord blood but also provide the placenta with the riate nutrients, including oxygen. The placenta may be cultivated and perfused with a similar solution which was used to remove the residual cord blood cells, preferably, without the addition of anticoagulant agents.

Stem cells can be ed from placenta by perfusion with a solution comprising one or more ses or other tissue-disruptive enzymes. In a specific embodiment, a placenta or portion thereof is brought to 25-3 7°C, and is incubated with one or more tissue-disruptive enzymes in 200 mL of a culture medium for 30 minutes. Cells from the perfusate are collected, brought to 4°C, and washed with a cold inhibitor mix comprising 5 mM EDTA, 2 mM dithiothreitol and 2 mM beta-mercaptoethanol. The placental stem cells are washed after several s with a cold (e.g., 4°C) stem cell collection composition bed elsewhere herein.

Perﬁlsion using the pan method, that is, whereby perﬁlsate is ted after it has exuded from the maternal side of the placenta, results in a mix of fetal and maternal cells. As a result, the cells collected by this method comprise a mixed population of placental stem cells of both fetal and maternal origin. In contrast, perfusion solely through the placental vasculature, whereby perfusion ﬂuid is passed through one or two placental vessels and is collected solely through the remaining vessel(s), results in the collection of a tion of placental stem cells almost exclusively of fetal origin. 545MW Stem cells from mammalian ta, whether obtained by perfusion or enyzmatic digestion, can initially be d from , be isolated from) other cells by Ficoll gradient centrifugation. Such centriﬁlgation can follow any standard protocol for centrifugation speed, etc. In one embodiment, for example, cells collected from the placenta are recovered from perfusate by centrifugation at 5000 x g for 15 minutes at room temperature, which separates cells from, e.g., contaminating debris and platelets. In another embodiment, placental perfusate is concentrated to about 200 ml, gently layered over Ficoll, and centrifuged at about 1100 x g for minutes at 22°C, and the low-density interface layer of cells is collected for further processing.

Cell s can be resuspended in fresh stem cell collection composition, or a medium suitable for stem cell maintenance, e.g., IMDM serum-free medium containing 2U/ml heparin and 2mM EDTA (GibcoBRL, NY). The total mononuclear cell fraction can be isolated, e. g., using prep (Nycomed Pharma, Oslo, Norway) according to the manufacturer’s recommended procedure.

As used herein, ting” tal stem cells means removing at least 20%, 3096,4096,5096,6096,7096,8096,9096,95960r99960ftheceﬂsxvﬁhvﬂnchtherﬂacmnalmeni cells are normally associated in the intact mammalian placenta.

Placental stem cells obtained by perfusion or digestion can, for example, be further, or initially, isolated by differential nization using, e.g., a solution of 0.05% trypsin with 0.2% EDTA (Sigma, St. Louis MO). Differential trypsinization is possible e placental stem cells typically detach from plastic surfaces within about ﬁve minutes whereas other nt populations typically require more than 20-30 minutes incubation. The detached placental stem cells can be ted following trypsinization and trypsin neutralization, using, e.g., Trypsin Neutralizing Solution (TNS, Cambrex).

In one embodiment of isolation of placental stem cells, aliquots of, for example, about 5-10 x 106 placental cells are placed in each of several T-75 ﬂasks, preferably f1bronectin- coated T75 ﬂasks. In such an embodiment, the cells can be cultured with commercially available Mesenchymal Stem Cell Growth Medium (MSCGM) ex), and placed in a tissue culture incubator (37°C, 5% C02). After 10 to 15 days, non-adherent cells are removed from the ﬂasks by washing with PBS. The PBS is then replaced by MSCGM. Flasks are preferably examined daily for the ce of various adherent cell types and in particular, for identiﬁcation and ion of clusters of ﬁbroblastoid cells.

The number and type of cells collected from a mammalian placenta can be monitored, for example, by ing changes in morphology and cell surface markers using standard cell ion techniques such as ﬂow cytometry, cell sorting, immunocytochemistry (e.g., staining with tissue speciﬁc or arker speciﬁc antibodies) ﬂuorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), by examination of the morphology of cells using light or confocal microscopy, and/or by measuring changes in gene expression using techniques well known in the art, such as PCR and gene expression proﬁling. These techniques can be used, too, to identify cells that are positive for one or more particular markers. For example, using antibodies to CD34, one can determine, using the techniques above, whether a cell comprises a detectable amount of CD34 as compared to, for example, an isotype control; if so, the cell is CD34+. se, if a cell produces enough OCT-4 RNA to be detectable by RT- PCR, or signiﬁcantly more OCT-4 RNA than a terminally-differentiated cell, the cell is OCT-4+.

Antibodies to cell surface markers (e.g., CD markers such as CD34) and the sequence of stem cell-speciﬁc genes, such as OCT-4, are well-known in the art.

Placental cells, particularly cells that have been isolated by Ficoll separation, differential nce, or a combination of both, may be sorted, e.g., further isolated, using a ﬂuorescence activated cell sorter (FACS). Fluorescence activated cell sorting (FACS) is a well- known method for separating particles, including cells, based on the ﬂuorescent properties of the particles (Kamarch, 1987, Methods Enzymol, 151:150-165). Laser excitation of ﬂuorescent moieties in the individual particles results in a small electrical charge allowing electromagnetic separation of positive and negative les from a e. In one embodiment, cell surface marker-speciﬁc antibodies or ligands are labeled with distinct ﬂuorescent labels. Cells are processed through the cell , allowing tion of cells based on their ability to bind to the dies used. FACS sorted particles may be directly deposited into individual wells of 96- well or 384-well plates to tate separation and cloning.

In one sorting scheme, placental stem cells can be sorted on the basis of expression of the s CD34, CD38, CD44, CD45, CD73, CD105, OCT-4 and/or HLA-G, or any of the other markers listed elsewhere herein. This can be accomplished in connection with procedures to select stem cells on the basis of their adherence properties in culture. For example, adherence selection of placental stem cells can be accomplished before or after sorting on the basis of marker expression. In one embodiment, for example, placental stem cells can be sorted ﬁrst on the basis of their expression of CD34; CD34’ cells are retained, and cells that are CD200+ or HLA-Gl, are separated from all other CD34’ cells. In another embodiment, placental stem cells can be sorted based on their expression of CD200 and/or HLA-G, or lack thereof; for example, cells displaying either of these markers can be isolated for r use. Cells that express, e.g., CD200 and/or HLA-G can, in a speciﬁc embodiment, be further sorted based on their expression of CD73 and/or CD105, or epitopes recognized by antibodies SH2, SH3 or SH4, or lack of expression of CD34, CD38 or CD45. For example, in one embodiment, placental stem cells are sorted by expression, or lack thereof, of CD200, HLA-G, CD73, CD105, CD34, CD38 and CD45, and placental stem cells that are CD200+, HLA-G’, CD73+, CD105+, CD347, CD3 8’ and CD45’ are isolated from other placental cells for further use.

In another embodiment, magnetic beads can be used to separate cells, e.g., separate placental stem cells from other placental cells. The cells may be sorted using a magnetic ted cell sorting (MACS) technique, a method for separating particles based on their y to bind magnetic beads 00 um diameter). A variety of useful ations can be performed on the magnetic microspheres, including covalent addition of antibody that speciﬁcally recognizes a particular cell surface le or hapten. The beads are then mixed with the cells to allow binding. Cells are then passed through a magnetic ﬁeld to separate out cells having the c cell surface marker. In one embodiment, these cells can then isolated and ed with magnetic beads coupled to an antibody against additional cell surface markers. The cells are again passed through a magnetic ﬁeld, ing cells that bound both the antibodies. Such cells can then be diluted into separate dishes, such as microtiter dishes for clonal isolation.

Placental stem cells can also be characterized and/or sorted based on cell morphology and growth characteristics. For example, tal stem cells can be characterized as having, and/or selected on the basis of, e.g., a ﬁbroblastoid appearance in culture. Placental stem cells can also be characterized as having, and/or be ed, on the basis of their y to form embryoid-like bodies. In one embodiment, for example, tal cells that are ﬁbroblastoid in shape, express CD73 and CD105, and produce one or more embryoid-like bodies in culture can be isolated from other placental cells. In another embodiment, OCT-4+ placental cells that produce one or more embryoid-like bodies in culture are isolated from other tal cells.

In another embodiment, placental stem cells can be identiﬁed and terized by a colony forming unit assay. Colony forming unit assays are commonly known in the art, such as Mesen CultTM medium (Stem Cell Technologies, Inc., Vancouver British Columbia).

Placental stem cells can be assessed for viability, proliferation ial, and longevity using standard techniques known in the art, such as trypan blue exclusion assay, ﬂuorescein diacetate uptake assay, propidium iodide uptake assay (to assess viability); and thymidine uptake assay, MTT cell proliferation assay (to assess proliferation). Longevity may be determined by methods well known in the art, such as by determining the maximum number of population doubling in an extended culture.

Placental stem cells can also be separated from other placental cells using other techniques known in the art, e.g., selective growth of desired cells (positive selection), selective destruction of ed cells (negative ion); separation based upon differential cell agglutinability in the mixed population as, for example, with soybean agglutinin; freeze-thaw procedures; filtration; conventional and zonal centriﬁJgation; centrifugal elutriation (counter- streaming centrifugation); unit gravity separation; countercurrent bution; electrophoresis; and the like. .5 CULTURE OF PLACENTAL STEM CELLS .5.1 Culture Media Placental stem cells, including the arPSCs bed herein, can be cultured in any medium, and under any conditions, recognized in the art as acceptable for the culture of stem cells. In n embodiments, the culture medium comprises serum. In certain ments, placental stem cells, including the asPSCs described herein, can be cultured in, for e, DMEM-LG (Dulbecco’s Modiﬁed ial Medium, low glucose)/MCDB 201 (chick last basal medium) containing ITS (insulin-transferrin-selenium), LA+BSA (linoleic acid-bovine serum n), dextrose, L-ascorbic acid, PDGF, EGF, IGF-l, and penicillin/streptomycin; DMEM-HG (high glucose) comprising 10% fetal bovine serum (FBS); DMEM-HG comprising % FBS; IMDM (Iscove’s modif1ed Dulbecco’s medium) comprising 10% FBS, 10% horse serum, and hydrocortisone; M199 comprising 10% FBS, EGF, and heparin; u-MEM (minimal essential ) comprising 10% FBS, GlutaMAXTM and gentamicin; DMEM comprising 10% FBS, GlutaMAXTM and gentamicin, etc. A preferred medium is DMEM-LG/MCDB-201 comprising 2% FBS, ITS, LA+BSA, dextrose, L-ascorbic acid, PDGF, EGF, and penicillin/streptomycin.

Other media in that can be used to culture placental stem cells, including the asPSCs described herein, include DMEM (high or low e), Eagle's basal medium, Ham's F10 medium (F10), Ham's F-12 medium (F12), Iscove's modiﬁed Dulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGM), Liebovitz's L-15 medium, MCDB, DMIEM/F12, RPMI 1640, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma), and CELL- GRO FREE.

The culture medium can be supplemented with one or more components including, for example, serum (e.g., fetal bovine serum (FBS), preferably about 2-15% (v/v); equine (horse) serum (ES); human serum (HS)); beta-mercaptoethanol (BME), preferably about 0.001% (v/v); one or more growth factors, for e, platelet-derived growth factor , mal growth factor (EGF), basic fibroblast grth factor (bFGF), insulin-like growth -1 (IGF-l), leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), and erythropoietin (EPO); amino acids, including L-valine; and one or more otic and/or antimycotic agents to control microbial contamination, such as, for example, penicillin G, streptomycin sulfate, amphotericin B, gentamicin, and in, either alone or in ation. .5.2 Expansion and Proliferation of Placental Stem Cells Once placental stem cells, including the asPSCs described herein, are isolated (e.g., separated from at least 50% of the placental cells with which the stem cell or population of stem cells is normally associated in viva), the stem cells or population of stem cells can be proliferated and expanded in vitro. For example, once anoikis resistant tal stem cells are produced, such cells can also be proliferated and ed in vitro. Placental stem cells, including the asPSCs described herein, can be cultured in tissue culture containers, e.g., dishes, ﬂasks, multiwell plates, or the like, for a sufficient time for the placental stem cells to proliferate to 70-90% conﬂuence, that is, until the placental stem cells and their progeny occupy 70-90% of the culturing surface area of the tissue culture container. tal stem cells, including the asPSCs described herein, can be seeded in culture vessels at a density that allows cell growth. For e, the placental stem cells may be seeded at low density (e.g., about 1,000 to about 5,000 cells/cmz) to high density (e.g., about 50,000 or more cells/cmz). In a preferred embodiment, the placental stem cells are cultured at about 0 to about 5 percent by volume C02 in air. In some preferred ments, the tal stem cells are cultured at about 2 to about 25 percent 02 in air, preferably about 5 to about 20 t 02 in air. The placental stem cells preferably are cultured at about 25°C to about 40°C, preferably 37°C. The placental stem cells are ably cultured in an incubator. The culture medium can be static or agitated, for example, using a bioreactor. Placental stem cells can be grown under low oxidative stress (e.g., with addition of glutathione, ascorbic acid, catalase, tocopherol, N-acetylcysteine, or the like).

Once 70%-90% conﬂuence is obtained, the placental stem cells, including the asPSCs described herein, may be passaged. For example, the cells can be enzymatically treated, e.g., trypsinized, using techniques well-known in the art, to separate them from the tissue culture surface. After removing the placental stem cells by pipetting and counting the cells, about -100,000 stem cells, preferably about 50,000 tal stem cells, are passaged to a new culture container containing fresh culture medium. Typically, the new medium is the same type ofmedium from which the stem cells were removed. Provided herein are populations of placental stem cells, including the asPSCs described herein, that have been passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, l2, l4, 16, 18, or 20 times, or more, and combinations ofthe same. .6 PRESERVATION OF ANOIKIS RESISTANT PLACENTAL CELLS Anoikis ant placental stem cells can be preserved, that is, placed under conditions that allow for long-term storage, or conditions that t cell death by, e.g., apoptosis or necrosis.

Anoikis ant placental stem cells can be preserved using, e.g., a composition comprising an sis inhibitor, necrosis inhibitor and/or an oxygen-carrying perﬂuorocarbon, as described in related US. Provisional ation No. 60/754,969, entitled “Improved Composition for Collecting and Preserving Placental Cells and Methods of Using the Composition” ﬁled on December 25, 2005.

In one embodiment, provided herein is a method of preserving anoikis resistant placental stem cells comprising contacting said anoikis resistant placental stem cells with a stem cell collection composition comprising an inhibitor of apoptosis and an oxygen-carrying perﬁuorocarbon, wherein said inhibitor of apoptosis is present in an amount and for a time sufﬁcient to reduce or prevent apoptosis in the population of anoikis resistant placental stem cells, as compared to a population of anoikis resistant placental stem cells not ted with the inhibitor of apoptosis. In a speciﬁc embodiment, said inhibitor of apoptosis is a caspase inhibitor. In another speciﬁc embodiment, said tor of apoptosis is a INK inhibitor. In a more speciﬁc embodiment, said JNK inhibitor does not modulate differentiation or proliferation of said anoikis resistant placental stem cells. In another embodiment, said stem cell collection composition comprises said inhibitor of sis and said oxygen-carrying perﬂuorocarbon in separate phases. In another embodiment, said stem cell collection composition comprises said inhibitor of sis and said oxygen-carrying perﬁuorocarbon in an emulsion. In r ment, the stem cell collection ition additionally comprises an emulsiﬁer, e.g., lecithin. In another embodiment, said apoptosis inhibitor and said perﬂuorocarbon are between about 0°C and about 25°C at the time of contacting the stem cells. In another more speciﬁc embodiment, said apoptosis inhibitor and said perﬂuorocarbon are between about 2°C and 10°C, or between about 2°C and about 5°C, at the time of contacting the stem cells. In r more speciﬁc embodiment, said contacting is performed during transport of said anoikis resistant placental stem cells. In another more speciﬁc embodiment, said contacting is performed during freezing and thawing of said population of anoikis ant placental stem cells.

In another ment, anoikis resistant placental stem cells can be preserved by a method comprising contacting said anoikis resistant placental stem cells with an inhibitor of apoptosis and an organ-preserving compound, n said inhibitor of apoptosis is present in an amount and for a time sufﬁcient to reduce or prevent apoptosis of the s resistant tal stem cells, as compared to anoikis resistant placental stem cells not contacted with the inhibitor of apoptosis. In a speciﬁc embodiment, the organ-preserving compound is UW solution (described in US. Patent No. 4,798,824; also known as n; see also Southard et al., Transplantation 49(2):25 1-257 ) or a solution described in Stern et al., US. Patent No. 267. In another embodiment, said organ-preserving compound is hydroxyethyl starch, lactobionic acid, rafﬁnose, or a combination thereof In another embodiment, placental stem cells, to be used to produce s resistant placental stem cells, are contacted with a stem cell collection composition comprising an apoptosis inhibitor and oxygen-carrying perﬂuorocarbon, organ-preserving compound, or combination thereof, during perfusion. In another embodiment, said placental stem cells, to be used to produce anoikis resistant placental stem cells, are contacted during a process of tissue disruption, e.g., enzymatic digestion. In another embodiment, placental cells, to be used to produce anoikis resistant placental stem cells, are contacted with said stem cell collection nd after collection by perﬁJsion, or after collection by tissue disruption, e.g., enzymatic digestion.

Typically, during placental stem cell collection, enrichment and isolation, it is preferable to minimize or eliminate cell stress due to hypoxia and ical stress. In another embodiment of the method, therefore, placental stem cells, to be used to produce s resistant placental stem cells, are d to a hypoxic condition during collection, enrichment or isolation for less than six hours during said preservation, wherein a hypoxic ion is a concentration of oxygen that is less than normal blood oxygen concentration. In a more speciﬁc embodiment, said placental stem cells are exposed to said hypoxic condition for less than two hours during said vation. In another more speciﬁc embodiment, said placental stem cells are exposed to said hypoxic condition for less than one hour, or less than thirty minutes, or is not exposed to a hypoxic ion, during collection, enrichment or isolation. In r speciﬁc embodiment, said tal stem cells are not exposed to shear stress during collection, enrichment or isolation.

The s resistant placental stem cells, as well as the placental stem cells to be used to produce anoikis resistant placental stem cells, described herein can be cryopreserved, e. g., in cryopreservation medium in small containers, e.g., es. Suitable cryopreservation medium includes, but is not limited to, culture medium including, e.g., growth medium, or cell freezing medium, for example commercially available cell freezing medium, e.g., C2695, C2639 or C6039 (Sigma). eservation medium ably comprises DMSO (dimethylsulfoxide), at a tration of, e.g., about 10% (v/v). Cryopreservation medium may comprise additional agents, for example, Plasmalyte, methylcellulose with or t glycerol. The stem cells are preferably cooled at about l°C/min during cryopreservation. A preferred cryopreservation temperature is about -80°C to about -l80°C, preferably about -125°C to about -l40°C.

Cryopreserved cells 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. eserved cells preferably are thawed at a temperature of about 25°C to about 40°C, preferably to a temperature of about 37°C. In certain embodiments, anoikis ant placental stem cells provided herein are cryopreserved about 12, 24, 36, 48, 60 or 72 hours after being contacted with modulatory RNA molecules (e.g., transfection). In one embodiment, anoikis resistant placental stem cells provided herein are cryopreserved about 24 hours after being contacted with modulatory RNA molecules (e.g., transfection). .7 COMPOSITIONS .7.1 Compositions Comprising Anoikis Resistant Placental Stem Cells ed herein are compositions sing the anoikis resistant placental stem cells described herein. Such compositions may comprise populations of anoikis resistant placental stem cells ed herein combined with any physiologically-acceptable or medically- acceptable compound, composition or device for use in, e.g., research or therapeutics. .7.1-1W The s resistant tal stem cells described herein can be preserved, for example, cryopreserved for later use. Methods for cryopreservation of cells, such as stem cells, are well known in the art. Anoikis resistant placental stem cells can be prepared in a form that is easily administrable to an individual. For example, anoikis resistant placental stem cells described herein can be contained within a container that is suitable for medical use. Such a container can be, for e, a sterile plastic bag, ﬂask, jar, vial, or other container from which the placental cell population can be easily dispensed. For example, the container can be a blood bag or other plastic, medically-acceptable bag le for the intravenous administration of a liquid to a recipient. The container is preferably one that allows for cryopreservation of the anoikis resistant placental stem cells.

Cryopreserved anoikis resistant placental stem cell populations can comprise anoikis resistant placental stem cells derived from a single donor, or from multiple donors. The anoikis resistant placental stem cells can be completely HLA-matched to an ed recipient, or partially or completely HLA-mismatched.

Thus, in one embodiment, provided herein is a composition comprising anoikis resistant tal stem cells in a container. In a speciﬁc ment, the anoikis resistant placental stem cells cryopreserved. In another speciﬁc embodiment, the container is a bag, ﬂask, vial or jar. In more speciﬁc embodiment, said bag is a sterile c bag. In a more speciﬁc embodiment, said bag is suitable for, allows or facilitates intravenous administration of said anoikis resistant placental stem cells. The bag can comprise multiple lumens or compartments that are onnected to allow mixing of the anoikis resistant placental stem cells and one or more other solutions, e.g., a drug, prior to, or during, administration. In another speciﬁc embodiment, the composition comprises one or more compounds that facilitate cryopreservation of the combined stem cell population. In another speciﬁc embodiment, said anoikis resistant tal stem cells are contained Within a physiologically-acceptable aqueous on. In a more speciﬁc embodiment, said logically-acceptable aqueous on is a 0.9% NaCl solution. In another speciﬁc embodiment, said anoikis resistant placental stem cells are HLA- matched to a ent of said anoikis resistant placental stem cells. In another speciﬁc embodiment, said anoikis resistant placental stem cells are at least partially HLA-mismatched to a recipient of said anoikis resistant placental stem cells. In r speciﬁc embodiment, said anoikis resistant placental stem cells are derived from placental stem cells from a plurality of donors. .7.1.2 Pharmaceutical Compositions In another aspect, provided herein is a pharmaceutical composition for treating an dual having or at risk of developing a disease, disorder or condition having an inﬂammatory component, said pharmaceutical ition comprising a therapeutically effective amount of anoikis resistant placental stem cells.

The anoikis resistant placental stem cells provided herein can be formulated into pharmaceutical compositions for use in viva. Such pharmaceutical compositions can comprise anoikis resistant placental stem cells in a pharmaceutically-acceptable carrier, e.g., a saline solution or other ed physiologically-acceptable solution for in viva administration.

Pharmaceutical compositions provided herein can comprise any of the s resistant placental stem cells described herein. The pharmaceutical itions can comprise fetal, al, or both fetal and maternal anoikis resistant placental stem cells. The pharmaceutical compositions provided herein can further comprise anoikis resistant placental stem cells produced from placental stem cells obtained from a single individual or placenta, or from a plurality of indiViduals or placentae.

The pharmaceutical compositions provided herein can comprise any number of anoikis resistant placental stem cells. For example, a single unit dose of anoikis resistant placental stem cells can comprise, in various embodiments, about, at least, or no more than 1 x 105, 5 x105,lx106,5 x106, 1x107,5 x107, 1x108,5 x108,lx109,5 x109, 1x1010,5 x1010, l x 1011 or more anoikis resistant tal stem cells.

The pharmaceutical compositions provided herein can comprise populations of anoikis ant tal stem cells that comprise 50% viable anoikis resistant placental stem cells or more (that is, at least 50% of the cells in the tion are ﬁanctional or living).

Preferably, at least 60% of the cells in the population are Viable. More preferably, at least 70%, 80%, 90%, 95%, or 99% of the anoikis resistant placental stem cells in the population in the pharmaceutical composition are Viable. 513WW Further provided herein are matrices, els, scaffolds, and the like that comprise anoikis resistant placental stem cells. The anoikis resistant placental stem cells provided herein can be seeded onto a natural matrix, e. g., a tal erial such as an amniotic membrane material. Such an amniotic membrane material can be, e.g., amniotic membrane dissected directly from a mammalian placenta; ﬁxed or heat-treated amniotic membrane, substantially dry (z'.e., <20% H20) amniotic membrane, chorionic membrane, substantially dry chorionic membrane, substantially dry amniotic and chorionic membrane, and the like. Preferred tal biomaterials on which anoikis resistant placental stem cells can be seeded are described in Hariri, US. Application Publication No. 2004/0048796.

The anoikis ant tal stem cells provided herein can be suspended in a hydrogel solution suitable for, e.g., ion. le hydrogels for such compositions include self-assembling peptides, such as RADl6. s resistant placental stem cells can also be combined with, e.g., alginate or platelet-rich , or other ﬁbrin-containing matrices, for local injection. In one embodiment, a hydrogel solution comprising anoikis resistant placental stem cells can be allowed to , for instance in a mold, to form a matrix haVing the cells dispersed therein for implantation. Anoikis resistant placental stem cells in such a matrix can also be cultured so that the cells are mitotically ed prior to implantation. The hydrogel can be, e. g., an organic polymer (natural or synthetic) that is cross-linked via covalent, ionic, or en bonds to create a three-dimensional open-lattice structure that s water molecules to form a gel. Hydrogel-forming materials include polysaccharides such as alginate and salts thereof, peptides, polyphosphazines, and polyacrylates, which are crosslinked ionically, or block polymers such as hylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature or pH, respectively. In some embodiments, the hydrogel or matrix is biodegradable.

In some embodiments, the matrix comprises an in situ polymerizable gel (see., e. g., US. Patent Application ation 2002/0022676; Anseth et al., J. Control Release, 78(1- -209 (2002); Wang et al., Biomaterz'als, :3969-80 (2003).

In some embodiments, the polymers are at least partially soluble in aqueous solutions, such as water, ed 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), poly(acrylic acids), poly(methacrylic , copolymers of acrylic acid and methacrylic acid, poly(vinyl acetate), and sulfonated polymers, such as sulfonated polystyrene. Copolymers having acidic side groups formed by reaction of acrylic or rylic acid and vinyl ether monomers or polymers can also be used.

Examples of acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably ﬂuorinated) alcohol groups, ic OH , and acidic OH groups.

The anoikis resistant placental stem cells can be seeded onto a three-dimensional framework or scaffold and implanted in viva. Such a framework can be implanted in combination with any one or more growth factors, cells, drugs or other components that stimulate tissue formation or otherwise enhance or improve the practice of the methods of treatment described elsewhere herein.

Examples of scaffolds that can be used herein include nonwoven mats, porous foams, or self assembling peptides. Nonwoven mats can be formed using ﬁbers comprised of a synthetic absorbable copolymer of glycolic and lactic acids (e.g., PGA/PLA) (VICRYL, n, Inc., Somerville, NJ). Foams, composed of, e.g., poly(s—caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer, formed by ses such as freeze-drying, or lyophilization (see, e.g., US. Pat. No. 6,355,699), can also be used as scaffolds.

In another embodiment, the scaffold is, or comprises, a nanoflbrous scaffold, e.g., an electrospun nanof1brous ld. In a more speciﬁc embodiment, said nanof1brous scaffold comprises poly(L-lactic acid) (PLLA), type I collagen, a mer of vinylidene ﬂuoride and roethylnee (PVDF-TrFE), poly(-caprolactone), poly(L-lactide-co-s—caprolactone) [P(LLA- CL)] (e. g., 75 :25), and/or a copolymer of poly(3 -hydroxybutyrate-cohydroxyvalerate) (PHBV) and type I collagen. Methods of producing nanof1brous scaffolds, e.g., electrospun nanof1brous scaffolds, are known in the art. See, e. g., Xu et (1]., Tissue ering 10(7): l 160- 1168 (2004); Xu et al., Biomaterials 25:877-886 (20040; Meng et al., J. Biomaterials Sci, Polymer Edition 18(1):81-94 (2007).

The anoikis resistant placental stem cells described herein can also be seeded onto, or ted with, a logically-acceptable ceramic material ing, but not limited to, mono-, di-, tri-, alpha-tri-, beta-tri-, and tetra-calcium phosphate, hydroxyapatite, ﬂuoroapatites, calcium sulfates, calcium ﬂuorides, calcium oxides, m carbonates, magnesium calcium phosphates, biologically active glasses such as BIOGLASS®, and mixtures thereof. Porous biocompatible ceramic materials currently commercially ble include SURGIBONE® (CanMedica Corp., Canada), ENDOBON® (Merck Biomaterial France, France), CEROS® s, AG, Bettlach, Switzerland), and mineralized collagen bone grafting products such as HEALOSTM (DePuy, Inc., Raynham, MA) and VITOSS®, RHAKOSSTM, and CORToss® (Orthovita, Malvem, Pa.). The ork can be a mixture, blend or composite of natural and/or synthetic materials.

In another embodiment, anoikis resistant placental stem cells can be seeded onto, or contacted with, a felt, which can be, e.g., composed of a multifilament yarn made from a bioabsorbable material such as PGA, PLA, PCL copolymers or blends, or hyaluronic acid.

The anoikis resistant placental stem cells bed herein can, in another embodiment, be seeded onto foam scaffolds that may be composite structures. Such foam scaffolds can be molded into a useful shape. In some embodiments, the ork is treated, e. g., with 0.1M acetic acid followed by incubation in polylysine, PBS, and/or collagen, prior to inoculation of the anoikis resistant tal stem cells 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 on of one or more proteins (e.g., collagens, elastic ﬁbers, reticular ﬁbers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitinsulfate, chondroitinsulfate, dermatan 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-thrombogenic. These treatments and materials may also 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 nt membrane ns such as laminin and Type IV collagen, synthetic materials such as EPTFE, and segmented polyurethaneurea silicones, such as NTM (The Polymer Technology Group, Inc., Berkeley, Calif). The scaffold can also comprise anti-thrombotic agents such as heparin; the lds can also be treated to alter the e charge (e.g., coating with plasma) prior to seeding with anoikis resistant placental stem cells. 6. EXAMPLES 6.1 EXAMPLE 1: IDENTIFICATION OF ANOIKIS ASSOCIATED GENES IN PLACENTAL STEM CELLS The existence of anoikis associated genes in placental stem cells was determined using the DecodeTM RNAi Viral ing Library (Thermo Scientiﬁc) in accordance with manufacturer’s ctions. Brieﬂy, the assay utilizes RNAi-based lentiviral logy to incorporate shRNAmirs into the genes of the target host cell genome. Cells are transduced with the shRNAmirs, and can be selected for by cell sorting based on the expression of green cent protein (GFP) by the shRNAmirs or using a puromycin assay (because the shRNAmirs n a gene that confers cin resistance to transduced cells). Selective pressure is then applied to identify cells that survive the pressure, and thus express certain genes at increased or decreased levels as a survival phenotype. Such differentially expressed genes are identiﬁed by PCR ampliﬁcation of the genomic DNA of the surviving cells, n the sequences of the shRNAmirs incorporated into speciﬁc genes (and that thus inhibit/downregulate the sion of those genes) are ampliﬁed. Accordingly, the speciﬁc genes implicated in conferring the survival phenotype can be identiﬁed.

An anoikis assay for placental stem cells was ﬁrst developed. It was determined that a suitable anoikis assay for placental stem cells that fulﬁlled the goal of having greater than 90% ofunmodiﬁed placental stem cells dead or apoptotic as ed to the control unmodiﬁed placental stem cells (cultured under attachment conditions) consisted of the following: g of placental stem cells at a concentration of 1 x 105 cells/ml in DMEM supplemented with 0.1% FBS and culturing the cells at 37°C, 5% C02, for 48-72 hours on a control plate (which allows cell attachment) or on a low-attachment plates selected from Corning Ultra-Low Attachment, Nunc Hydrocell, or Nunc Low Cell g. Figure 1 demonstrates that unmodiﬁed placental stem cells exhibit very low survival under low attachment conditions after 48 hours of culture, whereas equivalent s of unmodiﬁed placental stem cells demonstrate near 100% survival under assay ions that allow cell attachment. As shown in Figure 2, cpopy ed that the placental stem cells cultured under attachment conditions ng CellBind plates) were viable and demonstrated morphology characteristic of placental stem cells after 72 hours of culture, s as the placental stem cells cultured under low-attachment conditions (Corning Ultra-Low Attachment plates) failed to survive after 72 hours of culture under comparable culture conditions (the exception being the attachment conditions).

The established placental stem cell s assay was used as the selective pressure in the DecodeTM RNAi Viral Screening Library (Thermo iﬁc). Brieﬂy, tal stem cells were transduced with the Decode Viral Library at a MOI of 0.3 in serum-free DMEM with Polybrene according to the instructions of the manufacturer. Transduced cells were selected for using a FACS Aria (Becton Dickinson) cell sorter using GFP as the selectable marker. Next, the transduced placental stem cells were ted to the optimized anoikis assay described above for selection of anoikis-resistant placental stem cells. Surviving cells (e.g., anoikis resistant placental stem cells) after 48-hours of culture in the anoikis assay were isolated by either single cell sorting (using FACS) or serial dilution of GFP+ cells. The isolated cells were expanded in 384-well plates to reach >500 cells per well. Figure 3 depicts wells comprising populations of expanded placental stem cells identiﬁed in the assay (the bright-colored markings in the well represent GFP positive cells). The gene sion proﬁles from 187 wells of cells (wells with strong GFP expression) were assessed to identify s associated genes by isolating genomic DNA from the cells and subsequently PCR amplifying the barcode-containing fragments to facilitate sequence-based target gene identiﬁcation performed. The anoikis associated genes se those that were inhibited/downregulated in the ing cells and which thus were identiﬁed as being associated with the anoikis pathway in the placental stem cells.

Seventy-three genes were ﬁed as haVing a role in placental stem cell anoikis, ing the following genes: AMIGOl (NCBI GENE ID NO:57463); ARHGAP20 (NCBI GENE ID 69); CD38 (NCBI GENE ID NO:952); CLCCl (NCBI GENE ID NO:23155); CNTF (NCBI GENE ID NO:1270); ZFP91-CNTF (NCBI GENE ID NO:386607); COX8A (NCBI GENE ID NO:1351); DHX34 (NCBI GENE ID NO:9704); FAM175A (NCBI GENE ID NO:NO 51023); MRPSlSC (NCBI GENE ID NO:84142); FAM44C (NCBI GENE ID NO:284257); FBP2 (NCBI GENE ID NO:8789); FLIl (NCBI GENE ID NO:2313); FREM3 (NCBI GENE ID NO: 166752); IFIT5 (NCBI GENE ID NO:24138); LOC399851 (NCBI GENE ID NO:399851); LOC400713 (NCBI GENE ID NO:400713); LOC651610 (NCBI GENE ID NO:651610); PIGP (NCBI GENE ID NO:51227); SH3TC2 (NCBI GENE ID 28); SLC2A3 (NCBI GENE ID NO:6515); STAU2 (NCBI GENE ID NO:27067) TMEFFl (NCBI GENE ID NO:8577); TMEM217 (NCBI GENE ID NO:221468); TMEM79 (NCBI GENE ID NO:84283);USHBP1 (NCBI GENE ID NO:83878);APH1B (NCBI GENE ID NO:83464); ATP2B2 (NCBI GENE ID NO:491); C13orf39 (NCBI GENE ID NO: 196541); C4orf17 (NCBI GENE ID NO:84103); C4orf46 (NCBI GENE ID NO:201725); DDX41 (NCBI GENE ID NO:51428); DKFZp547J222 (NCBI GENE ID NO:84237); FGFRl (NCBI GENE ID NO:2260); FHDCl (NCBI GENE ID NO:85462); GNAI2 (NCBI GENE ID NO:2771); GP5 (NCBI GENE ID NO:2814); ILlRN (NCBI GENE ID NO:3557); KIF24 (NCBI GENE ID NO:347240); KNDCl (NCBI GENE ID NO:85442); LOC100132598 (NCBI GENE ID NO: 100132598); LOC151760 (NCBI GENE ID NO:151760); LOC152024 (NCBI GENE ID NO: 152024); LOC339833 (NCBI GENE ID NO:339833); LPAR4 (NCBI GENE ID 6); LSGl (NCBI GENE ID NO:55341); MAP3K5 (NCBI GENE ID NO:4217); PDK3 (NCBI GENE ID NO:5165); PELI2 (NCBI GENE ID NO:57161); RNF103 (NCBI GENE ID NO:7844); SNX31 (NCBI GENE ID NO: 169166); TXN2 (NCBI GENE ID NO:25828); and XKR7 (NCBI GENE ID NO:343702).

This Example demonstrates that placental stem cells o anoikis in low ment conditions and that speciﬁc placental stem cell genes that cause anoikis in placental stem cells (anoikis associated genes) exist. 2013/074892 6.2 EXAMPLE 2: GENERATION OF S RESISTANT PLACENTAL STEM CELLS Selected anoikis associated genes identiﬁed in Example 1 were targeted in placental stem cells using siRNA directed to the particular genes of interest. Placental stem cells were transfected using Dharmacon ON-TARGETplus SMARTpool siRNA c to selected genes at a ﬁnal siRNA concentration of 25nM, with Dharmafect l transfection reagent. Gene expression was analyzed using quantitative real-time PCR analysis was performed using 7900HT Fast Real-Time PCR System with TaqMan® Gene sion kits to examine gene silencing efﬁciency.

Once it was conﬁrmed that the siRNA speciﬁc to selected anoikis associated genes effectively inhibited/downregulated the expression of such genes, placental stem cells in which s ated genes were targeted were cultured in the anoikis assay bed in Example 1. The viability of these placental stem cells was assessed using the CellTiter AQueous One Solution Cell Proliferation Assay (MTS) and the CyQuant Direct assay, to determine whether anoikis ant placental stem cells could be generated by speciﬁcally ing anoikis associated genes in placental stem cells.

Figure 4 depicts the results of an MTS assay, wherein selected anoikis ated genes identiﬁed in Example 1 were inhibited/downregulated in placental stem cells using siRNA speciﬁc to the genes. The placental stem cells were subjected the anoikis assay described in Example 1 for 48 hours, and the viability of such cells was determined and ed to the viability of unmodiﬁed placental stem cells (placental stem cells not contacted with an siRNA speciﬁc to an anoikis associated gene; “Non-treated”) and placental stem cells that were contacted with non-targeting pool siRNA (“NTP”), which is not speciﬁc to any of the anoikis associated genes ﬁed herein.

As shown in Figure 4, the targeting of numerous of the anoikis associated genes identiﬁed in Example 1 resulted in sed viability of placental stem cells as compared to the non-treated and NTP placental stem cell groups (in all cases, placental stem cells targeted with anoikis associated gene-speciﬁc siRNA demonstrated increased viability relative to the NTP placental stem cell group). The placental stem cells that exhibit increased ity following targeting of anoikis associated genes represent anoikis resistant placental stem cells s), based on their increased ability to survive in tachment conditions as compared to unmodiﬁed placental stem cells. The CyQuant Direct viability assay veriﬁed that, under comparable conditions as the MTS assay, arPSCs could be generated by targeting s ated genes in placental stem cells (Figure 5).

Further analyses were performed on selected anoikis associated genes, the inhibition of which in placental stem cells resulted in signiﬁcant ses in placental stem cell viability in the anoikis assay (i.e., in low attachment conditions). In particular, the s of inhibition of the following anoikis associated genes were r assessed: FH2 domain containing 1 (FHDCl: NCBI GENE ID NO:85462), guanine nucleotide g protein alpha inhibiting 2 (GNAIZ; NCBI GENE ID NO:277l), kinase non-catalytic C- lobe domain containing 1 (KNDCl; NCBI GENE ID NO:85442), lysophosphatidic acid receptor 4 (LPAR4; NCBI GENE ID NO:2846), mitogen-activated protein kinase kinase kinase 5 (MAP3K5; NCBI GENE ID NO:4217), solute carrier family 2, member 3 (SLC2A3; NCBI GENE ID NO:6515), and staufen homolog 2 (STAU2; NCBI GENE ID NO:27067).

The CyQuant Direct viability assay conﬁrmed that, after culturing for 48 hours in the anoikis assay described above, arPSCs could be generated by targeting anoikis associated genes in placental stem cells (Figure 6). The inhibition/downregulation of each anoikis associated gene assayed resulted in increased ability of the placental stem cells to survive in low- attachment conditions as compared to placental stem cells targeted with non-speciﬁc siRNA (NTP), with inhibition/downregulation of ﬁve of the seven genes tested resulting statistically signiﬁcant increases in survival of the placental stem cells, conﬁrming that the placental stem cells had become resistant to s.

To further conﬁrm viability of the anoikis ant stem cells, an arPSC tion wherein solute carrier family 2, member 3 (SLC2A3; NCBI GENE ID NO:6515) was ted/downregulated, and an equivalent amount of unmodiﬁed placental stem cells were separately cultured for 3 days under low attachment conditions. After the three day culture period, the two cell populations were visualized using microscopy. Figure 7 demonstrates that higher numbers of anoikis resistant tal stem cells remained viable after the culture period (Figure 7A) as compared to the number of viable unmodiﬁed placental stem cells (Figure 7B).

This Example demonstrates that tal stem cells can be made resistant to anoikis by targeting particular anoikis associated genes in the placental stem cells using approaches that modulate the expression of the anoikis associated genes, including ing such genes with siRNA. The arPSCs generated in this Example can be advantageously used as therapeutics based on the fact that they do not require a substrate to adhere to in order to remain viable in vivo (for example, after systemic or local administration to a subject) and also may be ageously used in the large-scale propagation of placental stem cells as suspension es.

Equivalents: The compositions and methods disclosed herein are not to be d in scope by the speciﬁc embodiments described herein. Indeed, s modiﬁcations of the compositions and methods in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying ﬁgures. Such modiﬁcations are intended to fall within the scope of the appended claims.

Various publications, patents and patent applications are cited herein, the sures of which are incorporated by reference in their entireties.

Claims

WHAT WE CLAIM IS:

1. An isolated placental stem cell, wherein said placental stem cell is resistant to s, wherein said placental stem cell expresses at least one anoikis associated gene at a decreased level as compared to the sion of the same anoikis associated gene in an unmodified placental stem cell, and wherein said s ated gene is FHDC1 (NCBI GENE ID NO:85462), ), GNAI2 (NCBI GENE ID NO:2771), KNDC1 (NCBI GENE ID 42), LPAR4 (NCBI GENE ID 6), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), or STAU2 (NCBI GENE ID NO:27067).

2. The isolated placental stem cell of claim 1, n said anoikis resistant placental stem cell is a CD10+, CD34-, CD105+, CD200+ placental stem cell.

3. An isolated population of cells comprising anoikis resistant placental stem cells wherein said anoikis resistant placental stem cells express at least one anoikis associated gene at a decreased level as compared to the expression of the same anoikis ated gene in an unmodified placental stem cell, and n said anoikis associated gene is FHDC1 (NCBI GENE ID NO:85462), ), GNAI2 (NCBI GENE ID NO:2771), KNDC1 (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), or STAU2 (NCBI GENE ID NO:27067).

4. The isolated population of cells of claim 3, wherein said anoikis resistant placental stem cell is a CD10+, CD34-, CD105+, CD200+ placental stem cell.

5. The ed population of cells of claim 3 or 4, wherein at least 50% of the cells in said population of cells are anoikis resistant placental stem cells.

6. The isolated population of cells of claim 3 or 4, wherein at least 60%, at least 70%, at least 75%, at least 80%, and least 85%, at least 90%, at least 95%, or at least 99% of the cells in said population of cells are anoikis resistant placental stem cells.

7. A method of producing anoikis resistant placental stem cells comprising contacting placental stem cells with an effective amount of modulatory RNA molecules, such that said placental stem cells, after having been contacted with said modulatory RNA molecules express at least one anoikis associated gene at a decreased level as compared to the expression of the same anoikis associated gene in an equivalent amount of placental stem cells not contacted with said modulatory RNA les, wherein said anoikis associated gene is FHDC1 (NCBI GENE ID NO:85462), ), GNAI2 (NCBI GENE ID NO:2771), KNDC1 (NCBI GENE ID NO:85442), LPAR4 (NCBI GENE ID NO:2846), MAP3K5 (NCBI GENE ID NO:4217), SLC2A3 (NCBI GENE ID NO:6515), or STAU2 (NCBI GENE ID NO:27067), and provided said contacting is not within a human.

8. The method of claim 7, wherein said s ant placental stem cells survive in a low-attachment nment for a longer duration of time than an equivalent amount of placental stem cells not ted with said modulatory RNA molecules.

9. The method of claim 7 or 8, wherein said anoikis resistant placental stem cell is a CD10+, CD34-, CD105+, CD200+ placental stem cell.

10. The method of any one of claims 7 to 9, wherein said modulatory RNA molecules comprise small interfering RNAs (siRNAs), microRNA inhibitors (miR inhibitors), micro RNA mimics (miR mimics), antisense RNAs, short hairpin RNAs (shRNAs), or any combinations f.

11. The method of any one of claims 7 to 10, wherein said modulatory RNA molecules target at least one anoikis associated gene of said placental stem cells.

12. A composition comprising the isolated anoikis resistant tal stem cell of claim 1.

13. An isolated placental stem cell ing to claim 1, substantially as herein described or exemplified.

14. An isolated population of cells according to claim 3, substantially as herein described or exemplified.

15. A method according to claim 7, substantially as herein described or exemplified.