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PCX 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
WUKUU "^'^^iniemauonaJ Bureau 




..^.^.^^^^^ ^CATTON PUBUSHBD UNDER P .TBNT COOPHRAT.ON^;^E^ 



(51) International Patent Classification ^ : 

A61K 35/12, 35/28, 48/00, C12N 5/00 



A2 



(11) International Publication Number: 
(43) International Publication Date: 



WO 00/29002 

25 May 2000 (25.05.00) 



(21) InternaUonal Application Number: 



PCT/US99/26927 



nternational Filing Date: 12 November 1999 (12.1 1.99) 



(22) Int 



(30) Priority Data: 
60/108.357 



13 November 1998 (13.1 1.98) US 



nu Anolicant (for all designated States except US): OSIRIS 
(71) AppU^t^o^^^ INC. lUS/US); 2001 Aliceanna Street. 

Baltimorc. MD 21231-2001 (US). 
19010 (US). 

Aoents- ULLIE. Raymond et al.; Carella. Byrne. Bain. 
(74) Ag^?^- j^^Ji^^j S^^^rt & Olstein. 6 Becker Farm Road. 

Roseland. NI 07068 (US). 



(81) Designated States: AL. AM, ^T. AU Ba bB. BG^ BR 
RY CA CH CN. CR. CU. CZ. DE. DK.. DM, EE, ES. 1-1, 
II: Sd, GE. GH. GM. Hb, ID, IL. IS. JP. KE. KG. KP, 
KR KZ LC LK LR, LS, LT, LU, LV, MA, MD, MG, 
MK. MN.^ >ix. NO, NZ. PL. PT. RO.^JU. SD. SE. 
qr, SI SK. SU TJ. TM. TR. TT, TZ, UA. UG, US, UA 
tN. YU^ ZW, 'aRWO patent (GH, GM KE^ i^.^, 
SD, SU SZ, TZ. UG. ZW). Eurasian patent (AM. AZ, BY, 
KG, KZ. MD. RU. TJ. TM). European pat«it (AT. BE. CH, 
CY DE DK. ES. Fl. FR. GB. GR. IE. IT, LU. MC. NL, 
5?' st oS^l patent (BF. BJ. CF. CG. CI. CM. GA. ON. 
GW, ML, MR. NE. SN. TD. TG). 



Published i„x^,^,tom2/ search report and to be republished 

upon receipt of that report. 



I (54) Tiae: IN UTERO -mANSPLANTATION OF HUMAN MESE^^CHYMAL S11.M 
transplantation. 



RKicsnnnin <rWO 0029002A2 I > 



FOR THE PURPOSES OF INFORMATION ONLY 

cod.. \^ ■« -^y ^ «. per o. e» f~n, p.8« p-p^.» »«'"'°" ""'^ 



AL Albania 

AM Armenia 

AT Austria 

AU Austraiia 

AZ Azerbaijan 

BA Bosnia and Hcrxegovina 

BB Baibados 

BE Belgium 

BF Burkina Faso 

BG Bulgaria 

BJ Benin 

BR Brazil 

BY Bclanis 

CA Canada 

CF Central African Republic 

CG Congo 

CH Switzerland 

CI C6ie d'lvoirc 

CM Cameroon 

CN China' 

CU Cuba 

CZ Czech Republic 

DE Germany 

DK Dcnmaric 

EE Estonia 



ES Spain 

Fl Finland 

FR France 

GA Gabon 

GB United Kingdom 

GE Georgia 

GH Ghana 
ON Guinea 
GR Greece 
HU Hungary 
■ IE Ireland 
IL Israel 
IS Iceland 
IT Italy 
JP Japan 
KE Kenya 
KG Kyrgyzstan 
KP Democratic People's 

Republic of Korea 
KR Republic of Korea 
KZ Kazaksian 
LC Saint Lucia 
U Liechtenstein 
LK Sri Lanka 
LR Ubcria 



LS 


Lesotho 


LT 


Lithuania 


LU 


Luxembourg . 


LV 


Latvia 


MC 


Monaco 


MD 


Republic of Moldova 


MG 


Madagascar 


MK 


The fonner Yugoslav 




Republic of Macedonia 


ML 


Mali 


MN 


Mongolia 


MR 


Mauritania 


MW 


Malawi 


MX 


Mexico 


NE 


Niger 


NL 


Netherlands 


NO 


Norway 


NZ 


New Zealand 


PL 


Poland 


FT 


Portugal 


RO 


Romania 


RU 


Russian Federation 


SD 


Sudan 


SE 


Sweden 


SG 


Singapore 



SI 


Slovenia 


SK 


. Slovakia 


SN 


Senegal 


sz 


Swauland 


TD 


Chad 


TG 


Togo 


TJ 


Tajikistan 


TM 


Tuikmcnistan 


TR 


Turkey 


TT 


Trmidad and Tobago 


UA 


Ukraine 


VG 


Uganda 


US 


United States of America 


uz. 


Uzbekistan 


VN 


Viet Nam 


YU 


Yugoslavia 


ZW 


Zimbabwe 



BNSDOCID: <WO_OOe9002A2_l_> 



PCT/US99/26927 

WO"00/29002 



/N C^£i?0 TRANSPLANTATION OF 
HUMAN MESENCHYMAL STEM CELLS 

10 

ms application claims priority based on provisional application Serial No. 
60/108,357, filed November 13, 1998. 

He present invention relates to ft= field of cell therapy, and more 
,5 panicularly to the field ofin vivo gene therapy by administering naesenehyrnal stem 

cells. 

Background of the Invention 
Certain diseases, including inherited metabolic diseases, may produce 
20 irreversible damage to the fetus before birth. 

in utero hematopoietic stem cell transplantation is a potendal therapy for a 
large number of immunodeficiency diseases, hemoglobinopathies and o.h«s. 
Advanuges for the fetal recipient include potential immmiologic tolerance for 
25 transplanted cells, and mpid expartsion of the hematopoeitic compartment w.* 
fomtation of new "niches" for the competitive engraftment of donor cells. The 
existence of natural models of hematopoietic chimerism in dizygotic twins- whtch 
share cross placental circulation during development supports the potenual to 
achieve high levels of donor cell chimerism with prenatal transplantauon m 
30 recipients with normal hematopoiesis. 



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Experimental work in sheep and other animal models has[successfuliy 
achieved long-term multilineage allogeneic or xenogeneic hematopoietic chimerism 
in immimopermissive fetuses without the requirement for myeloablation or 
immunosuppression. This chimerism in immuno permissive fetuses has been shown 
5 to be secondary to the engraftment of true pluripotent hematopoietic stem cells. 

Limited clinical success has been achieved in immunodeficiency disorders in which 
there is a selective advantage for donor cells. However in most diseases this 
selective advantage does not exist and engraftment has been absent or low. Limited 
or lack of engraftment, both in terms of cell numbers and in terms of differentiated 
1 0 cell types, currently represents an obstacle to expanded clinical application of in 

utero stem cell transplantation. The requirement for transplantation during the brief 
period of immunopermissiveness is another obstacle of the establishment of a 
successful therapy. 

15 Mesenchymal stem cells are the formative pluripotential cells found inter 

alia in bone marrow, blood, dermis and periosteum that are capable of 
differentiation into any of the mesenchymal or connective tissues, for example, 
bone, cartilage, muscle, stroma, tendon, and fat. 

20 This homogeneous population of cells can be passaged in culture and may be 

characterized by the lack of hematopoietic cell markers and by the presence of a 
unique set of surface antigens. Under specific conditions they have been induced to 
form bone, cartilage, adipose tissue, tendon, and muscle, and in their 
undifferentiated state, resemble roughly stromal fibroblasts and can support 

25 hematopoiesis as evidenced by the support of LT-CIC in long term bone marrow 
culture. Preliminary in vivo studies suggest that these cells home to bone marrow of 
post-natal recipients after intravenous administration and can accelerate constitution 
after myeloablative conditioning regimes. 

30 Therefore it is an object of the invention to increase donor cell engraftment 

in fetal recipients. Another object is to regenerate damaged or diseased tissue by 
providing to a fetal recipient donor cells which can differentiate in situ. Yet another 
object is to prepare chimeric organs and tissues. Still another object is to treat a 

2 

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recipient by administering mesenchymal stem cells .wfficfi .Has? &en";hic«iifiHar:& 
express a therapeutic gene product. 

Summary of the Invention 

It has been discovered that mesenchymal stem cells (MSCs) transplanted into 
a fetus in utefo then were distributed throughout the fetus. The MSCs remained 
viable and differentiated into cellular types appropriate for the tissue or organ in 
which they engrafted. Surprisingly, the MSCs and their differentiated progeny were 
not rejected by immunocompetent hosts. The MSCs can be used for cellular therapy 

and tissue engineering. 

Normal MSCs express and secrete a number of cytokises, including G-CSF, 
SCF, LIF, M-CSF, IL-6, and IL-1 1- (Haynesworth, et al., J. Cell Physiol., Vol. 166, 
No. 3, pgs. 585-592 (March 1996)). As such, when functional non-self MSCs are 
transplanted into a fetus having defective autologeous MSCs. "gene therapy" thereby 
is performed on the host by virme of the delivery of said functional MSCs. 

It also has been discovered that when mesenchymal stem cells, which have 
been modified to cany exogenous genetic material of interest, are induced to 
differentiate, the progeny cells also carry the new genetic material. These 
transduced cells are able to express the exogenous gene product. Thus transduced 
mesenchymal stem cells and the cells differentiated therefrom can be used for 
applications where treatment using such modified ceUs is beneficial. For example, 
these modified cells can be used as a delivery system for therapeutic proteins 
encoded by the exogenous gene for treatment of inherited and/or acquired disorders 
25 of blood coagulation and wound healing. 

Accordingly, the present invention provides a method of obtaining 
genetically modified mesenchymal stem cells, comprising transducing mesenchymal 
stem cells with exogenous genetic material and placing the transduced cells under 
30 conditions suitable for differentiation of the mesenchymal stem cells into lineages 
which then contain the exogenous genetic material. 

Accordingly, the present invention is directed to a method for effecting gene 
therapy in vivo by administrating human mesenchymal stem cells in utero. The 



20 



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PCTAJS99/26927 



mesenchymal stem cells can differentiate into specificxell Imeages-Bepehding oii 
environment and integrate with like tissue to effect repair of tissue defects. 

In another embodiment of this aspect, the mesenchymal stem cells can be 
5 transduced with exogenous genetic material such that a gene product will be 
expressed by the mesenchymal stem cell or its differentiated progeny in vivo to 
provide a desired therapeutic effect. 

In another aspect, the present invention involves a method for treating a 
1 0 subject in need thereof by administering human mesenchymal stem cells in an 
amount effective to enhance the in vivo distribution and engraftment of 
mesenchymal stem cells. 

In another embodiment of this aspect, the mesenchymal stem cells can be 
15 transduced with exogenous genetic material such that a gene product will be 

expressed by the mesenchymal stem cell or its differentiated progeny in vivo to 
provide a desired therapeutic effect. 



20 



Brief Description of the Drawings 

Figure 1 is a photograph of liver tissue at 9 weeks after mesenchymal stem 
cells were injected intraperitoneally at 65 days gestation. 

Figure 2 is a photograph of bone marrow at 9 weeks after mesenchymal stem 
25 cells were injected intraperitoneally at 85 days gestation. 

Figure 3 is a photograph of heart tissue at 9 weeks after mesenchymal stem 
cells were injected intraperitoneally at 6i5 days gestation. 

30 Figure 4 is a photograph of thymus tissue at 9 weeks after mesenchymal 

stem cells were injected intraperitoneally at 85 days gestation. 



4 



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PCTAJS99/26927 

WO 00/29002 

Figure 5 shows results of PGR analysis for huinan specific P^2 rftitrogloMrl 
performed on tissue samples of liver (Lv), spleen (Sp), bone marrow (BM), thymus 
(Th). lung (Lg), brain (Br), and blood (Bd). 

Figure 6A is a photograph of a gel showing the presence of human B-2 
naicroglobulin DNA in liver, spleen, lung, bone marrow, thymus, brain, and blood 
isolated from sheep femses at 65 or 85 days gestation, wherein the sheep femses 
were g.vcn human mesenchymal stem cells in utero. Figures 6B, 6C, 6D, 6E, and 
6F arc photographs of slides showing the presence of human cells in sheep fetal 
liver, spleen, bone marrow, thymus, and lung, respectively. 

Figure 7A is a photograph of a slide showing human mesenchymal stem 
cells in the cardiac muscle of sheep fetuses stained with anti-human B-2 , 
„..croglobulin. Figures 7B and 7C are photographs of slides showing human cells xn 
the cardiac muscle of sheep fetuses stained with anti-human B-2 microglobuhn and 
SERCA-2. 

Figures 8 A and 8B are photographs of slides showing the presence of human 
B-2 microglobulin through nickel chloride staining in cartilage lacunae of lambs 
given human mesenchymal stem cells in ut^o at 65 days gestation, and wherem the 
cartilage was harvested at 2 months and 5 months after transplantation, respectively. 

Figure 9 shows photographs of slides of human cells, contacted with human- 
specific anti-CD23 antibody, found in the bone marrow of sheep at 5 months after ra 
utero transplantation of human mesenchymal stem cells. Figure 9A is a control 
;;;^g normal ovine.tissue contacted with human-specific anti-CD23 anubody. 
Figures 9B through 9D show CD23+ human cells in ovine bone marrow at 
increasing magnification. 

Figure 10 shows photographs of slides of human cells, contacted with 
human-specific anti-CD74 antibody, found in the bone marrow of sheep at 5 months 
after in ut^ transplantation of human mesenchymal stem cells. Figure lOA is a 
control showing normal ovine tissue contacted with human-specific ant.-CD74 



30 



WO-00/29002 



PCT/US99/26927 



antibody. Figures lOB through lOD show CD74+ hMriTi;eni.itii'uovine%ynias3t 
increasing magnification. 

Figures llA and IIB are photographs of slides showing human fi-2 
microglobulin positive cells in the central neivous system of sheep at 5 months after 
the sheep were given mesenchymal stem cells in utero . 



10 



15 



20 



25 



30 



Detailed Description of the Invention 

The human/sheep model is a unique model of widely disparate xenogeneic 
chimerism in which human hematopoietic stem cells survive for years in the sheep 
bone marrow after in utero transplantation. These cells can establish multilineage 
long term engraftment after retransplantation in utero into second generation 
recipients proving the engraftment of pluripotent hematopoietic stem cells. The 
system is limited by species specificity of hematopoietic cytokines. The sheep 
microenvironment can support the viability of human HSCs and progenitors but 
human cytokine administration is required to drive the cells toward differentiation 
and peripheral expression. Human cells can be detected in this system using a 
variety of sensitive methodologies including flow cytometry, fluorescence in situ 
hybridization, immunohistochemistry, and PGR. 

The present invention relates generally to the use of human , mesenchymal 
stem cells and to corhpbsitions comprising human mesenchymal stem cells for m 
utero administration. 

In order to obtain subject human mesenchymal stem cells for the methods 
described herein, mesenchymal stem cells can be recovered from other cells in the 
bone marrow or other mesenchymal stem cell source. (Pittenger, supra.) Bone marrow 
cells may be obtained from iliac crest, femora, tibiae, spine, rib or other medullary 
spaces. Other sources of human mesenchymal stem cells include embryonic yolk sac, 
placenta, umbiUcal cord, fetal and adolescent skin, and blood. The presence of 
mesenchymal stem cells in the culture colonies may be verified by specific cell 
surface markers which are identified with unique monoclonal antibodies, see, e.g.. 



BNSDOCID: <WO 0029002A2_I_> 



10 



15 



^ ^«««„n, PCT/US99/26927 
WO QO/29002 



U.S. Patent No. 5,486,359. These isolated mesenchimal reel! .jpSpulattcra^ 
epitopic characteristics associated only with mesenchymal stem cells, have the 
ability to regenerate in culture without differentiating, and have the ability to 
differentiate into specific mesenchymal lineages m v/rro onw vzvo. 

The mesenchymal stem cell populations can be autologous, allogeneic, or 
xenogeneic to the recipient. Preferably, mesenchymal stem cells are from the same 
species as the recipient. Most preferably, the MSCs are human in origin. 

Accordingly, any process that is useful to recover mesenchymal stem cells 
from human tissue may be utilized to result in a population of cells comprising 
mostly mesenchymal stem cells. In one aspect, a method of isolating mesenchymal 
stem cells comprises the steps of providing a tissue specimen containing 
mesenchymal stem cells, preferably bone marrow; isolating the mesenchymal stem 
cells from the specimen, for example by density gradient centrifugation; adding the 
isolated cells to a medium which contains factors that stimulate mesenchymal stem 
cell growth without differentiation and allows, when cultured, for the selective 
adherence of only the mesenchymal stem cells to a substrate surface; culturing the 
specimen-medium mixture; and removing the non-adherem matter from the 
20 substrate svirface. 

According to the method of the present invention, the isolated mesenchymal 
stem cells are culture expanded in appropriate media, i:e. cultured by methods which 
favor cell growth of the enriched cell populations. In general, the cells are plated^at 
25 a density of 0.05-2x10^ cells/cm^ preferably at a density of 0.5 -10 x 10^ cells/cm^ 

The cells may be characterized prior to, during, and after culture to 
detemiine the composition of the cell population, for example by flow cytometric 
analysis (FACS). The human mesenchymal stem cells can be stained with human 
30 mesenchymal stem cell-specific monoclonal antibodies. . 

The culmre conditions such as temperature, pH, and the like, are those 
previously used with the cells utilized in this invention and will be apparent to one 
of skill in the art. 



RNf^DOCID: -eWO 0029002A2 J..> 



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The mesenchymal stem cells produced according to the methods described 
herein can be used to provide a reliable and constant source of mesenchymal stem 
cells for individuals in need thereof, e.g. those in need of cellular therapy, tissue 
5 engineering or regeneration and gene therapy. 

Another aspect of the present invention relates to the introduction of genes 
into the mesenchymal stem cells such that the mesenchymal stem cells and progeny 
of the cells carry the new genetic material. 

10 

Thus, in accordance with this aspect of the invention, the mesenchymal stem 
cells can be modified with genetic material of interest. The mesenchymal stem cells ^ 
are able lo express the product of the gene expression and, with a signal sequence, 
secrete the expression product. These modified cells can then be administered to a 
15 target, i.e., in need of mesenchymal stem cells or the gene expression product, where 
the expressed product will have a beneficial effect. 

Thus, genes can be introduced into cells which are then returned to the 
autologous donor or provided to a non-self recipient where the expression of the 

20 gene will have a therapeutic effect. For example, mesenchymal stem cells may be 
genetically engineered to express therapeutic proteins. ^ Those that may be 
mentioned include providing continuous delivery of insulin, which at present must 
be isolated firom the pancreas and extensively purified or manufactured in vitro 
recombinantly and then injected into the body by those whose insulin production or 

25 utilization is impaired. Genetically engineered human mesenchymal stem cells can 
also be used for the production of clotting factors. Persons suffering firom 
hemophilia A lack a protein called Factor VIII, which is involved in clotting. A 
recombinant Factor VIII product is now available and is administered by injection 
(Kogenate®, Bayer, Berkeley, C A). Incorporation of genetic material- of interest into 

30 human stem cells and other types of cells is particularly valuable in the treatment of 
inherited and acquired disease. Inherited disorders that could be treated in this way 
include disorders of amino acid metabolism, such as Fabry's Disease, Gauchefs 
Disease, histidinurea or familial hypocholesterolemia; and disorders of nucleic acid 
. metabolism, such as hereditary orotic aciduris. Hvanan stem cells transduced with a 

8 

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gene encoding the missing or inadequately produc^Csubstahce : cairs'l^e^- .u^ 
produce it in sufficient quantities. For example, a Fabry's patient incapable of 
producing sufficient quantities of the enzyme alpha galactosidase A could be given 
MSCs transduced with the gene coding for that enzyme. 

5 

Some genetic diseases cause damage to the fetus or early post-natal 
individual; accordingly it is desirable to deliver the needed gene product in utero. 
Mesenchymal stem cells are advantageous particularly for gene therapy because 
they may be transduced with high efficiency, are long-lived and retain the ability to 
10 produce large numbers of daughter cells. Transduced MSCs express exogenous 
genes at high levels for long periods. This expression can continue through and after 
terminal differentiation. (Allay, et. al.. Human Gene Therapy , Vol. 8, No. 12, pgs. 
1417-1427 (August 10, 1997).) 

15 The mesenchymal stem cells may be genetically modified by incorporation 

of genetic material into the cells, for example using recombinant expression vectors. 

As used herein "recombinant expression vector" refers to a transcriptional 
unit comprising an assembly of (1) a genetic element or elements having a 

20 regulatory role in gene expression, for example, promoters or enhancers, (2) a 
structural or coding sequence which is transcribed into mRNA and translated into 
protein, and (3) appropriate transcription initiation and termination sequences. 
Structural units intended for use in eukaiyotic expression systems preferably include 
a leader sequence enabling extracellular Secretion of translated protein by a host ceU. 

25 AltOTiatively, where recombinant protein is expressed without a leader or transport 
sequence, it may include an N-terminal methionine residue. This residue may or 
may not be subsequently cleaved fi;om the expressed recombinant protein to provide 
a final product 

30 The mesenchymal stem cells thus may have stably integrated a recombinant 

transcriptional unit into chromosomal DNA or carry the recombinant transcriptional 
unit as a component of a resident plasmid. Cells may be engineered with a 
polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, for example. Cells 



9 



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may be engineered by procedures known in the art::ByIuse.of; a fetroviffl 
containing RNA encoding a polypeptide. 

Retroviruses from which the retroviral plasmid vectors hereinabove 
5 mentioned may be derived include, but are not limited to, Moloney Murine 
Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, 
Harvey Sarcoma Virus, avian leukosis vims, gibbon ape leukemia vims, human 
immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and 
mammary tumor virus. In one embodiment, the retroviral plasmid vector is MGIN, 
10 derived from murine embryonic stem cells. Generally regarding retroviral mediated 
gene transfer, see McLachlin et al.(1990). 

A preferred retroviral packaging cell line is described in U.S. Patent No. 
5,910,434, the contents of which are incorporated herein by reference. Such cell line 
15 permits very high levels of transfection, z.e., greater than 80%. 

The nucleic acid sequence encoding the polypeptide is under the control of a 
suitable promoter. Suitable promoters which may be employed include, but are not 
limited to, TRAP promoter, adenoviral promoters, such as the adenoviral major late 

20 promoter; the c>l;omegalovirus (CMV) promoter; the respiratory syncytial virus 
(RSV) promoter; inducible promoters, such as the MMT promoter, the 
metallothionein promoter; heat shock promoters; the albtimin promoter; the ApoAI 
promoter; human globin promoters; viral thymidine kinase promoters, such as the 
Herpes Simplex thymidine kinase promoter, retroviral long terminal repeats (LTRs); 

25 ITRs; the p-actin promoter; and hiiman growth hormone promoters; the GPIIb 
promoter. The promoter also may be the native promoter that controls the gene 
encoding the polypeptide. These vectors also make it possible to regulate the 
production of the polypeptide by the engineered progenitor cells. The selection of a 
suitable promoter will be apparent to those skilled in the art. The retroviral LTR is 

30 preferred. * 

It is also possible to use vehicles other than retroviruses to genetically 
engineer or modify the mesenchymal stem cells. Genetic information of interest can 

10 

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1 

wo 00/29002 PCT/US99/26927 



be introduced by means of any virus which can exprKOfie new^genetit ma 

such cells. For example, SV40, herpes virus, adenovirus, adeno-associated virus, 

and human papillomavirus can be used for this purpose. 

5 In addition, the expression vectors may contain one or more selectable 

iharicer genes to provide a phenotypic trait for selection of transformed cells such as 
dihydrofolate reductase, neomycin resistance or green fluorescent protein (GFP). 

The mesenchymal stem cells may be transfected through other means known 
10 in the art. Such means include, but are not limited to transfection mediated by 
calcium phosphate or DEAE-dextran; transfection mediated by the polycation 
Polybrene (Kawai and Nishizawa 1984; Chaney et al. 1986); protoplast fusion 
(Robert de Saint Vincent et al. 1981; Schaffoer 1980; Rassoulzadegan et al. 1982); 
electroporation (Neumann et al. 1982; Zimmermann 1982; Boggs et al. 1986); 
15 liposomes (see, e.g. Mannino and Gould-Fogerite (1988)). either through 
encapsulation of DNA or RNA within liposomes, followed by fiision of the 
liposomes with the cell membrane or, DNA coated with a synthetic cationic lipid 
can be introduced into cells by fiision (Feigner et al. (1987); Feigner and Hohn 
1989; Maurer 1989). 

20 

The present invention further makes it possible to genetically engineer 
mesenchymal stem cells in such a manner that they produce, in vitro or. in vivo 
polypeptides, hormones and proteins not normally produced in human mesenchymal 
stem cells in biologically significant amounts or produced in small amounts but in 

25 situations in which increased expression would lead to a tiierapeutic benefit. These 
products then would be secreted into the surrounding media or purified fix)m the 
cells. The human mesenchymal stein cells formed in this^ way can serve as 
continuous short-term or long-term production systems of the expressed substance. 
Alternatively, the cells could be modified such that a protein normally expressed 

30 will be expressed at much lower levels. This can be accomplished with antisense 
nucleic acid technology, catalytic enzyme expression, single chain antibody 
expression, and the like. 



11 



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This technology may be used to produce additibftal EopiesSf esseiiifial geixeS 
to allow augmented expression by the mesenchymal stem cells of certain gene 
products in vivo. These genes can be, for example, hormones, matrix proteins, cell 
membrane proteins, cytokines, adhesion molecules, "rebuilding" proteins important 
5 in tissue repair. The expression of the exogenous genetic material in vivo^ is often 
referred to as "gene therapy". Disease states and procedures for which such 
treatments have application include genetic disorders and diseases of blood and the 
immune system. Cell delivery of the transformed cells may be effected using 
various methods and includes intravenous or intraperitoneal infusion and direct 
1 0 depot injection into periosteal, bone marrow and subcutaneous sites. 

The mesenchymal stem cells of the present invention may be administered to 
the fetus using methods generally known in the art. 

15 In another aspect, the present invention provides a method of modifying fetal 

organs of a first species by administering mesenchymal stem cells of a second 
species to a fetus of the first species in utero. In a preferred embodiment, human 
mesenchymal stem cells are administered to a non-hxmian fetus in utero. Although 
the scope of this aspect of the present invention is not to be limited to any theoretical 

20 reasoning, it is believed that the non-human fetal organs may be less immimogenic, 
in the context of subsequent transplants into humans than unmodified organs. (See, 
for example, published PCT AppUcation No. W099/47163.) Thus, the modified 
non-htiman organs may be more suitable for transplantation. 

25 This aspect of the present invention is applicable particularly to the 

transplantation of animal organs into humans. Such organs include, but are not - 
limited to, the heart, pancreas, kidney, skin, liver, thymus, spleen, bone marrow, 
cartilage, and bone.' For example, human mesenchymal stem cells are administered 
to a non-human animal fetus. The MSCs preferably are autologous to the human 

30 patient. After birth, the non-human animal is raised to an appropriate age and/or 

size such that the size of the organ or organs to be transplanted approximates the size 
of the organ or organs required by the human. The modified organ(s) then is (are) 
harvested and transplanted into the hxmian patient. 

12 

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As an illustrative embodiment, mesenchymal stem cells feidm a pediatric; 
patient with a severe congenital heart defect are administered to a pig fetus. After 
birth, the pig would be raised to an appropriate age and/or size such that its heart 
approximates the size of the patient's heart. The modified heart then would be 
removed and transplanted into the pediatric patient. 

The above description of the invention and the following examples are by 
way of illustration only. Other permutations and practices of the invention will be 
readily envisioned by one of ordinary skill in the art by view of the above in 
conjunction with the appended drawings. Therefore, such permutations and 
variations are vdthin the scope of the present invention. 

EXAMPLE 1 

Human bone marrow aspirates routinely used for the isolation of the 
mesenchymal stem cells (MSCs), were purchased from Poietic Technologies, 
Gaithersburg, MD. Fresh bone marrow was obtained by routine iliac crest aspiration 
from normal human donors after informed . consent was obtained. MSCs were 
isolated as previously described. (Pittenger, et al.. Science , Vol. 284, pgs. 143-147 
(1999)). In brief, lOmL of bone marrow aspirate was added to 20mL of Control 
Media, Dulbeccos Modified Essential Media (Gibco/BRL, Gaithersburg, MD) 
containing 10% fetal bovine serum (Hyclone Inc., Logan, UT) from selected lots, 
and centrifuged, to pellet the cells and remove the fat. The cell pellet was 
resuspended in Control Media and fractionated on a density gradient generated by 
centrifiiging a 70% PercoU solution (supplier) at 13,000g for 20 minutes. The MSC 
enriched low density fraction was collected, rinsed with Control Media, and plated 
at 1 X lO' nucleated cells/60 mm^ dish! The MSCs were cultured in Control Media 
at 37°C in a humidified atmosphere containing 5% CO2. Upon reaching near- 
confluence, the cells were detached with 0.25% trypsin containing ImM EDTA 
(Gibco/BRL) for 5 minutes at 37*C. The cells were washed with Control Media and 
resuspended at 5 x lO^MSCs/mL in Control Media containing 5% DMSO (Sigma 
Chemical Co., St. Louis, MO). The cells were then stored in liquid nitrogen for use 
in the subsequent experiments. 



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To determine the ability of human mesenchymiaOtem cellS;^^^ 
to and engraft in hematopoietic sites and other tissues in the fetal sheep recipient, 
MSC*s were transplanted by intravenous or intraperitoneal injection into fetal sheep: 
5x10^ MSCs/fetus at 65 days gestation and 50x10^ MSCs/fetus at 85 days gestation 
5 (representing different ontologic stages of hematopoietic development in the sheep). 
At 50 days hematopoiesis is exclusively found in the fetal liver with no significant 
stroma detectable in the bone marrow. At 65 days the fetal liver remains the 
primary hematopoietic organ but stromal elements can be seen in the marrow with a 
few hematopoietic cells. At 80 days both fetal liver and bone marrow hematopoiesis 
1 0 are both active. In addition, the day 65 fetus does not mount an immune response to 
non-self hematopoietic cells, whereas the day 85 fetus is immune competent and 
rejects hematopoietic cell transplants routinely. 

Recipients were sacrificed at 7 or 14 days after transplantation and the liver, 
15 spleen, bone marrow, thymus, lung, brain and blood were analyzed by PGR for 
human specific p-2 microglobulin. Tissues positive for human sequence were 
confirmed by immunohistochemistry for morphologic assessment by staining for 
human P-2 microglobulin with secondary staining with horseradish peroxidase for 
visualization. 

20 

Methods 

Fetal Lamb Injections : 

Time dated pregnant ewes were sedated using ketamine and mask 
inJialational halothane. The animal was secured on the operating table in the supine 

25 position, endotracheally intubated, and mechanically ventilated. Anesthesia 
throughout the procedure was maintained with a halbthane/oxygen mixture titrated 
for adequate depth of sedation. An IV was started in the extemal jugular vein for 
infusion of lactated Ringer's solution and preoperative antibiotics. The ewe*s 
abdomen was sterilely prepared and draped. Using standard aseptic technique, a 

30 vertical midline laparotomy was performed to allow exposure of the uterus. Fetal 
lambs at 65 or 85 days gestational age underwent transuterine-intraperitoneal or 
intravascular injection of 5 x 10^ 50 x 10^ MSCs/fetus, respectively. For the group 
imdergoing intravascular injection, the ewes underwent horizontal hysterotomy with 



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5 



the use of electrocautery and Babcock clamps to control bleeding at tfie'^cut -uterus 
margin. The hind limbs and base of the umbilical cord were exteriorized and 
intravascular injection perforaied. The fetal hind limbs and umbilical cord were 
returned to the amniotic cavity. Amniotic fluid volume was restored with warm, 
sterile lactated Ringer's solution and the hysterotomy closed with a TA-90 stapler. 



The maternal abdomen was closed in layers and dressed with colloidin. The 
ewe was placed in a movable cage where she was monitored until completely 
recovered from anesthesia. The ewes were continuously monitored until completely 

10 alert, and were able to stand, eat and drink. At this time, the animal was transported 
back to her holding room, Buprenorphine was administered to alleviate 
postoperative pain. The animals were checked by the investigator's team 4-S hours 
later, and then once or twice daily. Further doses of buprenorphine were 
administered every 8 hours as needed for pain. Also, antibiotic (liquamycin) was 

15 given daily for 5 days. On each postoperative visit: the general well-being of the 
animal was assessed; the wound examined both visually and by palpation to detect 
signs of infection or dehiscence; and the vagina was examined externally to look for 
a discharge or mucous plug, both signs of preterm labor, 

20 Animals, including ewes and lambs, were euthanized at 7 or 14 days after 

transplantation via initial sedation using ketamine and then lethal, intracardiac KCl 
injection. Various tissues, including liver, lung, bone marrow, thymus, spleen, 
brain, and blood were harvested for histopathology and for ianalysis of the presence 
of human engraftment. For PGR analysis of tissues (cardiac muscle, thymus, bone 

25 marrow, muscle and spleen), animals were sacrificed at 9 weeks (4 animals 65 day, 
4 animals 85 day) (Figuresl-4). 

Tissue Processing : 

Fetal tissues were fixed overnight in 10% neutral buffered formalin at A^'C 
30 and paraffin embedded. For isolation of total cellular DNA, samples from each 
tissue were snap frozen in liquid nitrogen, and stored at -80°C for later DNA 
extraction. 



Immunohistochemistry : 

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Serial Sum sections were obtained from eSch ' 6f thfe^^ ^ 
tissues using a 30/50 microtome. Sections were deparaffinated, dehydrated, and 
rehydrated and then subjected to microwave antigen retrieval. Sections were then 
stained immunohistochemically for human class I antigen and SH2/SH3 antigen. 
5 The latter two antigens are foimd on MSCs. (See U.S. Patent No. 5,837,539.) 

Polymerase Chain Reaction : 

Total cellular DNA from the organs mentioned above were isolated using 
DNAzoI. Specific primers for human class I antigen were selected based on the 

10 published human class I sequence. In brief, lug DNA were added to each 0.65 mL 
microcentrifuge tube and placed on ice. A master mix was prepared and added on 
ice such that the final concentration of reagents for each sample was 2.5U Amplitaq 
Gold DNA polymerase (Perkin Elmer, Norwalk, CT). 200 uM deoxytriphosphates 
(dNTP's, Pharmacia, Piscataway, NJ), 50mM KCl, lOmM Tris-Cl (pH 8.3 at 22°C), 

15 1.5mM MgCh, 0.01% gelatin, and luM upstream and downstream primers. The 
-samples were kept on ice until the themiocycler block reached 94°C, when the 
samples were immediately placed into the block for 9 minutes. Samples were 
amplified for 40 cycles of 30 seconds at 94**C followed by 30 seconds of primer 
annealing followed by 1 minute of extension at 72°C. Upon completing the final 

20 cycle, samples were incubated for 5 minutes at 72°C. PGR products were subjected 
to electrophoresis through a 2.5% NuSieve/1% Seakem agarose gel containing 0.5ug 
ethidium bromide/mL in IX Tris acetate running buffer. The gels were illuminated 
with UV 280-nm light and photographed with type 55 positive/negative Polaroid 
film. The negative was scanned transmissively and the intensities of the bands 

25 determined using the Intelligent Quantifier densitometer. Band intensities was 
compared to standard curves generated with known concentrations of human DNA. 

EXAMPLE 2 

30 Animals: Time dated pregnant Westem Cross sheep carrying twin gestations 

(Thomas Morris, Reisterstown, MD) were housed in the ALAAC approved large 
animal facility at the Children's Hospital of Philadelphia and fed standard chow and 
water ad libitum. To determine the distribution and potential for differentiation of 

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human mesenchymal stem cells (MSG) following in ttm^ii^ccximi; 
human MSCs/kg estimated fetal weight were injected into the peritoneal cavity of 65 
or 85 day gestation fetal sheep. To determine the distribution of human MSCs 
following in utero injection, the fetal sheep were sacrificed at 1 or 2 weeks or 2 or 5 
months after injection and the liver, spleen, lung, bone marrow, thymus, brain, heart, 
skeletal muscle, cartilage, and blood were harvested and analyzed for the presence 
of human cells. In a small subset of 65 day gestation fetal sheep, the fetal tails were 
docked at the time of MSG injection and the tail wounds harvested at 1 week or 2 
months after wounding for DNA isolation and immunohistochemistry. 



10 



Tissue Processing: Fetal tissue harvested above were eitfier fixed overnight in 10% 
neutral buffered formalin (Fisher Scientific, Atlanta, GA) at 4°C. Bone marrow 
samples were then decalcified in Gal-EX (Fisher) for 12 hours, followed by a 3-4 
hour wash with distilled water. Samples were then paraffin embedded as previously 
1 5 described (Culling, Handbook of Histopathological and Histoc hemical Techniques, 
Butterworth Co., London (1974)). In addition, samples fi-om each tissue were snap 
frozen in liquid nitrogen, and stored at 80°G for subsequent total cellular DNA 
extraction. 

20 DNA Isolation: Total cellular DNA from the organs mentioned above was isolated 
using DNAzol (Molecular Resource Center, Inc., Cincinnati, OH). In brief, 
approximately 100 mg of tissue was homogenized in ImL of DNA zol. The DNA 
was precipitated with 0.5mL of 100% ethanol. The DNA precipitate was pelleted by 
centrifiigation and then washed twice with 95% ethanol. The DNA pellet was then 

25 dissolved in sterile water. 



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PCR Analysis: To screen the ovine tissues for the presence of human cells, total 
cellular DNA was subjected to PCR analysis for human specific B-2 microglobulin 
using a modification of previously described methods (Gilliland, et al., Proc. Nat, 
5 Acad. Sci, , Vol. 87, pgs. 2725-2729 (1990)). In brief, lug of total cellular DNA 
isolated fi-om the above-mentioned tissues was added to individual 0.65mL 
microcentrifiige tubes and placed on ice. A master mix was prepared and added on 
ice such that the final concentration of reagents for each sample was 2.5U Amplitaq 
Gold DNA polymerase (Perkin Elmer, Nonvalk, CT), 200uM deoxytriphosphates 
10 (dNTP's, Phannacia, Piscataway, NJ), 50mM KCl, lOmM Tris-Cl (pH 8.3 at 22°C), 
1 .5mM MgC 1 2, 0.0 1 % gelatin, and 1 uM upstream and downstream primers. 
Specific primers for human fi-2 microglobulin were selected based on the published 
human sequence (D), (upstream primer 5 -GTGTCTGGGTTTCATCAATC, 
downstream primer 5'-GGCAGGCATACTCATCTTIT) and shown to amplify 
is specifically human, not ovine, DNA. The samples were kept on ice until the 

thermocycler block reached 95 °C, when the samples were placed immediately into 
the block for 9 minutes. Samples were amplified for 50 cycles of 30 seconds at 
94''C followed by 30 seconds of primer annealing at SS^'C followed by 1 minute of 
extension at 72°C. Upon completing the final cycle, samples were incubated for 5 
20 minutes at 72°C. PCR products were subjected to electrophoresis through a 2.5% 
NuSieve/1% Seakem agarose gel containing 0.5ug ethidium bromide/mL in IX Tris 
acetate running buffer. The gels were illuminated with UV 280-nm light and 
photographed with type 55 positive/negative Polaroid film. 



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Immunohistochemistry: To verify the PGR results, i{ba5SofcM^ertltfS^--'fe3*: 
human fi-2 microglobulin was performed as previously described. To assess 
differentiation, immunohistochemical staining was also performed for human CD74, 
a marker of thymic epithelium (F), human CD23, a marker for bone mairow stroma 
5 (G), smooth endoplasmic reticulum calcium ATPase-2 (Serca-2), a marker for 
cardiac, skeletal, and smooth muscle (H), or glial fibrillary acid protein (GFAP), a 
marker for central nervous tissue (I). In brief, paraffin sections (4-5nm) were 
collected on Superfrost Plus slides (Fisher) from each of the paraffin embedded 
tissues. Slides were incubated for 24 hrs. at 55°C and then deparaffinated by 30 
10 min. immersion in xylene followed by rehydration through a graded alcohol series to 
deionized water over 1 0 min. and allowed to air dry completely. To enhance antigen 
retrieval, the slides were immersed in Tissue Unmasking Fluid (Ted Pella, Redding, 
CA). Blocking for 30 minutes at room temperature (RT) was performed using non- 
immune serum from the species in which the primary antibody was raised (1 :20 
1 5 dilution), followed by a 1 2 hour incubation with the specific primary antibody. The 
primary antibody dilutions used were as follows: human B-2 microglobulin 
(Pharmingen International, San Diego, CA, 1:200); human CD74 (Pharmingen, 
1:10); or human CD23 (Vector Laboratory, Burlingame, CA, 1:10). The slides were 
then washed with PBS followed by a second blocking step with methanol containing 
20 0.3% hydrogen peroxide for 30 minutes at room temperature. Slides were then 
rinsed with deionized water, then PBS, followed by incubation with biotinylated 
secondary antibody (1:200 dilution) for 30 min. at RT. The slides were washed with 
PBS and avidin-biotin complex added for 45 min. at RT. The sUdes were then 
rinsed well in PBS, developed with the chromagen 3,3'-diaminobenzidine. For 
25 sections stained for human B-2 microglobulin, CD74, and CD23 the sUdes then were 



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lightly counterstained with hematoxylin. For cardiac muscle and'l>'mn;^to^^^^ 
B-2 microglobulin was developed first using nickel chloride as the chromagen, and 
then subjected to a secondary immunohistochemical staining for SERCA-2 (Vector 
Laboratory', 1 :50 dilution) or GFAP (Vector Laboratory, 1 : 1 00 dilution), 
5 respectively, as previously described (Van Der Loos, et al., Histochem. J. , Vol. 25, 
pgs. 1-11 (1993); VanDer Loos, et al., J. Hist. Cyto. , Vol. 42, pgs. 289-294 (1994)). 
Secondary' staining was developed using Vector VIP substrate kit (Vector 
Laboraton'). No counterstaining was performed on these double-stained slides. 

10 Results 

PCR Assessment of Human MSC Distribution 

In order to assess the early distribution of himian MSCs following in utero 
transplantation, PCR for human specific B-2 microglobulin DNA sequences was 
performed on DNA isolated fi-om liver, spleen, lung, bone marrow, thymus, brain, 
heart, skeletal muscle, and blood fi-om fetuses transplanted at either 65 or 85 days 
gestation. Tissue was harvested at 2 weeks, 2 months, or 5 months after in utero 
transplantation. Two weeks after transplantation himian B-2 microglobulin DNA 
was detected in all tissues examined in fetal sheep transplanted at 65 and 85 days 
gestation (Figure 6 A and Table 1), with the exception of skeletal muscle. Cartilage 
was not examined. After 2 months, human DNA was still detected in all tissues 
examined from fetuses transplanted at 65 days gestation including cartilage, with the 
exception of brain. In fetuses transplanted at 85 days gestation, human DNA was 
detected after 2 months in the spleen, bone marrow, thymus, heart, and blood. Five 
months after in utero transplantation (3 months after birth), human DNA was 

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detected in the bone marroyv, thymus, spleen, lung, ckiti'lage, arid' Mood of fetuife^ 
transplanted at 65 days and in the heart, brain, skeletal muscle, and blood of fetuses 
transplanted at 85 days. Although the pattern of human cell distribution in 
individual animals differed, human specific sequences were detected in all animals 
5 transplanted at the time of sacrifice (Table 1 ). 

Immunohistochemical Assessment of Human MSG Distribution 

The presence of human cells in tissues positive by PGR was confirmed by 
immunohistochemistry, using an antibody specific for human B-2 microglobulin 
10 (Pharmingen, San Diego, GA, mouse IgM, Clone TU99) a component of the Class I 
antigen complex. Negative controls, consisting of tissues from transplanted sheep 
that were negative by PGR, and of matched tissues from normal sheep, confirmed 
the human specificity of the staining (data not shown). Many human MSGs were 
seen in pre- and post-natal hematopoietic and lymphopoietic tissues including the 
1 5 fetal liver, bone marrow, spleen, and thymus (Figures 6B-6E). Multiple human 

MSGS could often be appreciated in a single high-power field (Figures 6B and 6C) 
in these tissues. Human cells were also identified in non-lymphohematopoietic sites 
including the heart, skeletal muscle, cartilage, perivascular areas of the GNS, and 
lung (Figure 6F). Five months after transplantation, human cells continued to be 
20 present in multiple tissues including the bone marrow, thymus, cartilage, heart, 
skeletal muscle, and brain. 

Immunohistochemical Assessment of Human MSG Differentiation 

Differentiation of human MSGs in various tissues following transplantation 
25 was assessed by one of three techniques: 1) characteristic morphology on anti- 



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human S-2 microglobulin staining; 2) immunohistochemical double- staiiSin'g for 
anti-human B-2 microglobulin and a second non-human specific differentiation 
marker; or 3) when available, positive staining with human specific differentiation 
markers proven to not cross-react with sheep cells. Using these techniques, site- 
5 specific differentiation was confirmed for human cardiomyocytes, chondrocytes, 
bone marrow stromal cells, thymic stromal cells, and skeletal myocytes. Human 
cells were identified in the CNS. 

Cardiomyocyte Differentiation 

10 To assess differentiation of human MSCs found in cardiac muscle, double- 

staining immunohistochemistry was performed using anti-human 6-2 microglobulin 
and anti-SERCA-2 (Smooth Endoplasmic Reticulum ATPase). At 2 and 5 months 
after in utero transplantation, human cells were detected in the cardiac muscle of 
fetuses transplanted at 65 and 85 days gestation. These cells had similar 

15 morphology to the surrounding ovine cardiomyocytes and also double-stained with 
human B-2 microglobulin and SERCA-2, consistent with human cardiomyocyte 
differentiation (Figures 7B and 7C). 

Chondrocyte Differentiation 

20 Chondrocyte differentiation was identified by the finding of human B-2 

microglobulin positive cells in cartilage lacunae of lambs transplanted at 65 days 
and harvested at 2 months or 5 months after transplantation. Immunohistochemistry 
was performed using a nickel chloride-based developing technique giving the 
particulate appearance observed (Figures 8A and SB). The immunohistochemical 

25 identification of human cells within the lacimae of cartilage specimens that were 

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DNA PGR positive for human S-2 microglobulin sequences represents clear 
evidence of human chondrocyte differentiation. 

Bone Marrow Stromal Differentiation 

5 To assess differentiation of human MSCs foimd in the bone marrow 

immxmohistochemistry was performed using a human specific anti-CD23 antibody 
(Phaimingen, San Diego, CA, mouse Igd, Clone M-L233). CD23 is the low 
affinity IgE receptor and has been shown to be expressed on a variety of cell types 
including bone marrow stromal cells (Huang, et al.. Blood , Vol. 85, pgs. 3704-3712 

10 (1995); Fourcade, et al., European Cytokine Network , Vol. 3, pgs. 539-543 (1992)). 
At 5 months after in utero transplantation many human cells were seen in the 
marrow and were demonstrated to express CD23 (Figures 9B through 9D). These 
human CD23 positive cells appeared to be large cells clustered in areas with ovine 
hematopoietic elements, consistent with bone marrow stroma, 

15 

Thymic Stromal Differentiation 

To assess differentiation of human MSCs foxmd in the thymus, 
immxmohistochemistry was performed using a hiiman specific anti-CD74 antibody 
(Pharmingen, San Diego, CA, mouse IgGi, Clone LN2). At 5 months after in utero 

20 transplantation, multiple human cells were detected in the thymus that strongly 
expressed CD74 (Figures lOB through lOD), an MHC associated invariate chain 
expressed on thymic stromal cells (Schlossman, et al.. Leukocyte Typing V: White 
Cell Differentiation Antigens , Oxford University Press, New York (1995)). These 
cells were large and were similar in morphologic appearance to nearby ovine thymic 

25 epithelium. 



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Human Cell Persistence in the CNS 

To assess differentiation of human MSCs found in the brain, double-staining 
immunohistochemistry was performed using anti-human 6-2 microglobulin and anti- 
GFAP (Glial Fibrillary Acid Protein). At 5 months after in utero transplantation, 
numerous P-2 microglobulin positive human cells were detected on the surface of 
the brain in the perivascular areas within the giral sulci (Figure 1 1). The 
differentiation state of the cells was not determined. 

Assessment of Human MSG Participation In Tissue Repair after Wounding: 

To assess the possible participation of human MSCs in tissue repair after 
wounding, tail wounds were created in five 65 day gestation fetal sheep at the time 
of MSG injection. One animal was sacrificed at one week and four animals at 2 
months. Human li-2 microglobulin DNA was detected by PGR in the one tail 
wound at 1 week and in one of four tail wounds at 2 months. The PGR results were 
verified by human B-2 microglobulin iiiununihistochemistry (data not shown). The 
cells expressing himian B-2 microglobulin in the tail woxmd appeared in the dermis 
and dermal appendages and had the morphologic appearance of fibroblasts 
consistent with participation in the woimd healing response. 

Discussion 

Mesenchymal stem cells are of increasing interest to the emerging fields of 
tissue engineering, cellular transplantation, and gene therapy because of their 
availability in bone marrow, their relative ease of expansion in culture, their 
amenability to genetic manipulation, and most importantly, their capacity for 



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10 



differentiation into multiple mesenchymal tissues. THese properties support 
potential clinical applications of: 1 ) large scale tissue engineering particularly for 
repair of musculoskeletd injury; 2) cellular therapy for diseases of mesenchymal 
origin such as muscular dystrophy, osteoporosis, osteogenesis imperfecta, and 
collagen disorders; 3) bone marrow conditioning to facilitate engraftment of 
autologous or allogeneic hematopoietic stem cells; and 4) gene therapy. Prenatal 
MSG transplantation may provide a "reservoir" of normal stem cells to replace 
defective cells as they become damaged in degenerative diseases with progressive 
cellular and organ damage. 



Experiments utilizing porous diffusion chambers or ceramdc cubes have 
documented the capacity of MSCs to form fibrous tissue, cartilage, or bone in vivo 
nCadivala, et al.. Cell Transplantation , Vol. 6, pgs. 125-134 (1997)). In addition, 
MSCs have been shown to improve healing of segmental bone defects and cartilage 
15 defects following direct implantation into injury sites (Wakitani, et al., J. Bone& 

Joint Surg, - American Volume , Vol. 76, pgs. 579-592 (1994)), Of greater relevance 
to this study are studies that have followed the fate of MSCs or MSC like 
populations following intravenous or intraperitoneal transplantation. There have 
been two studies in mice in which cultured mouse adherent cell populations have 
20 been transplanted and documented to persist following transplantation. In the first, 
cells from transgenic mice expressing a human mini-gene for collagen I were used 
as mesenchymal progenitor donors and the fate of the cells followed after 
transplantation into irradiated mice (Pereira, et al., Proc. Nat. Acad. Sci. , Vol. 92, 
pgs. 4857-4861 (1995)), Donor cells were detected in bone marrow, spleen, bone 
25 cartilage, and lung up to 5 months later by PCR for the human mini-gene, and a PGR 



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in situ assay on lung indicated that the donor cells diffusely populated tne 
parenchyma. Reverse transcription-PCR assays indicated that the marker collagen I 
gene was expressed in a tissue-specific manner. A second study transplanted either 
cultured adherent cells or whole bone marrow into irradiated mice with a phenotype 
of fragile bones resembling osteogenesis imperfecta caused by expression of the 
human minigene for type I collagen (Pereira, et al., Proc. Nat. Acad. Sci. , Vol. 95, 
pgs. 1 142-1 147 (1998)). With either source of cells, a similar distribution of 
engraftment was documented as observed in the previous study and in addition, 
fluorescense in situ hybridization assays for the Y chromosome indicated that, after 
2.5 months, donor male cells accounted for 4-19% of the fibroblasts or fibroblast- 
like cells obtained in primary cultures of the lung, calvaria, cartilage, long bone, tail, 
and skin. 

Our study is the first to docimient directly multipotential differentiation of a 
relatively well characterized MSG population, in vivo, after transplantation. 

The results help define specific aspects of MSG transplant biology. First, 
MSGs, although very large cells, can be transplanted, and are capable of homing to 
and engrafting in multiple tissues, even when transplanted into the fetal peritoneal 
cavity. This requires the transplanted MSG to cross endothelial barriers, integrate 
into host tissue microenvironments, and survive with available growth factors and 
regulatory signals. Our findings of a variable pattern of long-term MSG 
engraftment, following initial engraftment in nearly all tissues studied, supports a 
model of non-selective homing with subsequent selective long-term survival in 
specific tissues. This may be a fimction of the ability of specific microenvironments 
to support the engraftment and differentiation of MSGs, or alternatively, the loss of 



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engraftment frdm some tissues may be due to heterogetifeityiol ttie transplanted 
population with respect to differentiation potential or replicative capacity. Immune 
mediated rejection is less likely since the pattern of engraftment was not limited to 
immune privileged sites, and an immune mechanism should result in eradication of 
5 donor cells. 

A second observation of this study is that MSCs are capable of site specific, 
multipotential differraitiation and tissue integration following transplantation. 
Human MSCs have been shown /« vitro to differaitiate into adipocytic, 
1 0 chondrocytic, or osteocytic lineages (Pittenger, supra). Less well characterized 
MSG populations from other species have been induced in vitro toward myocytic 
differoitiation. This study confirms in vivo chondrocytic differentiation and for the 
first time clearly demonstrates in vivo cardiomyocytic and myocytic differentiation 
of a defined human MSG population. MSCs derived from bone marrow from 
1 5 multiple species have been demonstrated to support hematopoiesis writh equal or 
greater efficacy than stromal layers formed in long term Dexter cultures. Our study 
supports the role of MSCs in stromal support of hematopoiesis, both in the fetal 
liver, and postnatal bone marrow. We found multiple large human cells intimately 
associated with clusters of hematopoiesis in the fetal liver at 2 and 9 weeks after 
20 transplantation. In addition, large cells that stained positively for human specific 
CD23, were identified in the bone marrow at 9 and 22 weeks after transplantation. 
CD23 has been identified as a low affinity IgE receptor as well as a fiinctional CD21 
ligand (Huang, et al., 1995; Aubry, et al.. Cell, Vol. 57, pgs 1073-1081 (1989)) 
present on a variety of hematopoietic cells as well as bone marrow stromal cells 
25 (Fourcade, et al., 1992). Our interpretation of CD23 positive cells in this study as 



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"stromal" is based on the large size of the cells and the well' documented absence bf 
human hematopoietic cells in either the donor cell population or the recipient bone 
marrow. 

5 A relatively surprising finding was the presence of large thymic cells that 

stained positive for human specific B-2 microglobulin and CD74. CD74 is a cell 
surface MHC class Il-associated invariant chain molecule that is expressed on B- 
cells, Langerhans cells, dendritic cells, activated T-cells, and thymic epithelium 
(Schlossman, et al., 1995). The morphology of CD74+ cells in this study appears 

1 0 similar to the ovine thymic epithelial cells in the surrounding thymus. The precursor 
of thymic dendritic cells is thought to be the hematopoietic stem cell, whereas the 
origin of the thymic epithelial cell is unknown. Our data support a mesenchymal 
origin for the thymic epithelial cell as a "stromal" supporting cell in the thymus. 
Finally, the presence of human fibroblast like cells in tail wound sites suggests that 

1 5 MSCs are capable of appropriate differentiation for participation in repair of 
damaged tissues. 

The persistence of human cells observed in this xenogeneic model, even 
when transplanted after the development of iiiimunocompetence in the sheep fetus is 

20 intriguing. Potential mechanisms for tolerance include failure of immune 

recognition, local immune suppression, or thymic deletional tolerance. Human 
MSCs are known to express Class I HLA antigen but do not express Class II, which 
may limit immune recognition. Although thymic stromal cells are known to 
participate in thymocyte positive and negative selection and host thymic antigen 

25 presenting cells are capable of facilitating clonal deletion of donor reactive 



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lymphocytes after in utero HSC transplantation (Kini^'fetali, J: Pediatr. Siiirg. , VSli; 
34, pgs. 726-730 (1999)), neither mechanism would account for tolerance after the 
appearance of mature lymphocytes in the peripheral circulation. In vitro^ MSCs 
added to mixed lymphocyte cultures, however, have been shown to non-specifically 
5 ablate alloreactivity by an as yet unknown mechanism (Schwartz, 1989; Sha, et al., 
1988; Kim, et al., 1999). It is believed that the persistence of MSCs in this model 
results from a combination of minimal immunogenicity, and local immune 
suppression. 

10 It is to be understood, however, that the scope of the present invention is not 

to be limtied to the specific embodiments described above. The invention may be 
practiced other than as particularly described and still be within the scope of the 
accompanying claims. 



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CLAIMS 

L A method for treating a fetus comprising administering mesenchymal stem 
cells to the fetus. 

5 

2. The method of Claim 1 wherein the mesenchymal stem cells differentiate in 
vivo. 

3. The method of Claim 1 wherein the mesenchymal stem cells are modified 
10 with exogenous genetic material. 

4. The method of Claim 1 wherein said fetus is non-human. 

5. The method of Claim 4 wherein said mesenchymal stem cells are human. 

15 

6. A method of engrafting mesenchymal stem cells, comprising: administering 
mesenchymal stem cells to a fetus in utero. 

7. The method of Claim 6 wherein said fetus is non-human. 

20 

8. The method of Claim 7 wherein said mesenchymal stem cells are himian, 

9. A method of preparing an organ for transplantation, comprising: 

25 (a) administering mesenchymal stem cells to a non-human fetus 

in utero ; and 

(b) harvesting said organ. 
30 1 0. The method of Claim 9 wherein said non-human fetus is ovine. 

11. The method of Claim 9 wherein said mesenchymal stem cells are human. 

12. The method of Claim 9 wherein said organ is harvested after the birth of said 
35 fetus. 

13. A method of xenotransplantation comprising: transplanting the organ of 
Claim 12 into a human patient, 

40 1 4. The method of Claim 1 3 wherein said organ is a heart. 

15. The method of Claim 1 3 wherein said organ is a pancreas. 

16. The method of Claim 1 3 wherein said organ is a kidney. 



45 



30 



BNSOOCID: <WO_0029002A2_L> 



10 



wo 00/29002 PCT/US99/26927 

1 7. The method of Claim 1 3 wherein said organ is'a^iiver. 

1 8. The method of Claim 13 wherein said organ is skin. 

1 9. The method of Claim 1 3 wherein said organ is a thymus. 

20. The method of Claim 13 wherein said organ is a spleen. 

2 1 The method of Claim 1 3 wherein said organ is bone marrow. 

22. The method of Claim 1 3 wherein said organ is cartilage. 

23. Th method of Claim 13 wherein said organ is bone. 



15 24. 



20 



A hybrid organ comprising an organ of an animal of a first species and 



mescnchNTnal stem cells from a second species. 

25. The hybrid organ of Claim 24 wherein the mesenchymal stem cells have 
differcniiatcd into cells of that organ. 

26. The hybrid organ of Claim 24 wherein said first species is non-human, and 
said second species is human. 

27. The hybrid organ of claim 24 wherein said organ of an animal of a first 
25 species is selected from heart, lung, kidney, pancreas, skin, liver, splera, thymus, 

bone, cartilage, and bone marrow. 



31 



wo 00/29002 



I / 10 



PCT/US99/26927 



Fl G. 1 




BNSDOCID: <WO 00e9002A2_l_> 



SUBSTITUTE SHEET (RULE 26) 



wo 00/29002 PCT/US99/26927 

2 / 10 



F I G. 2 



jr,. JV|SG Inject^cf IP, at 85 Days GGsfatfon 

^•v<^-«'f'^> J- /Bone Marrow at-'s Weeks post-lnjettlon 



€0 'i':;^' J" 



% 4 • 



»f • *^ - * * 



- W ''V-' 



SUBSTITUTE SHEET (RULE 26) 



wo 00/29002 



3/10 



PCT/US99/26927 



F l G. 3 



MSC Injectad IP at §5 Days GesUtfon 
Heart at 9 Weeks p08t-lnjectlon:V 



V 



BNSOCCID: <WO 0a29002A2J_> 



SUBSTITUTE SHEET (RULE 26) 



wo 00/29002 



4/ 10 



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F I G. 4 



v.^ « -r«n w * ■ Injected IP at 85 Days Gestation 

^ - ' Thymus at 9 Weeks post-Injection 




1^ 



^ 



♦4 




SUBSTITUTE SHEET (RULE 26) 



wo ffO/29002 



PCT/US99/26927 



5/10 

F 1 G. 5 






F 1 G . 7B 



F 1 G. 70 




BNSDOCID: <WO 0029002A2_I_> 



SUBSTITUTE SHEET (RULE 26) 



wo 00/29002 



PCT/US99/26927 



6 / 10 



F 1 G. 6 A 




ladder ^ 



#618-85 Day; 
IV inj . 



#821 -65Day; 
IP inj. 



to 



F 1 G. 6 B 



.Fetal Liver 




F I G. 6C 



Spl 




F 1 G. 6D 



Bone Marrow 




F 1 G.6 E 

Thymus 





wo 00/29002 



PCTAJS99/26927 



7 / 10 

F 1 G. 8A 




F 1 G. 8B 




BNSDOCIO: <WO 0029002A2_I_> 



SUBSTrrUTE SHEET (RULE 26) 



wo 00/29002 



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8 / 10 






F 1 G . 9 C 




F 1 G. 9 D 



SUBSTITUTE SHEET (RULE 26) 



wo 00/29002 PCT/US99/26927 

9/10 



F 1 G. lOA 




F I G. lOB 




Fl G.IOC F I G. lOD 




SUBSTrrUTE SHEET (RULE 26) 

BNSOOCIO; <WO. 0a29002A2_L> 



PCT/DS99/26927 

WO 00/29002 

1 0/ 10 



F 1 G. II A 





SUBSTITUTE SHEET (RULE 26) 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCX 

TNTRRNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 7 : 
A61K 35/12, 35/28, 48/00, AOIK 
67/027, C12N 5/00, 15/00 



A3 



(11) International Publication Number: 
(43) International Publication Date: 



WO 00/29002 

25 May 2000 (25.05.00) 



(21) International Application Number: PCTAJS99/26927 

(22) International Filing Date: 12 November 1999 (12.11.99) 



(30) Priority l>ata: 

60/108,357 



13 November 1998 (13,11.98) US 



f71^ ADplicanl thr ail designated States except US): OSIRIS 
THERAPHirriCS. INC. [US/US]; 2001 Aliceanna Street, 
Baltimore, MD 21231-2001 (US). 

I nS) KSjJican^ (for US only): THIEDE. Mark [USAJS]; 
I ^^^^ '"^572 MarVairr Dnvc, St. l^uis. MO 63146 (US). FLAKE, 
Alan [US/US I; 1131 Springmont Circle, Bryn Mawr, PA 
19010 (US). 

1 (74) Agents: LILLIE, Raymond el al.; Carella, Byrne. Bain 
Gilfillan, Cccchi, Siewan & Ol stein, 6 Becker Farm Road. 
Roseland, NJ 07068 (US). 



(81) Designated States: AL, AM. AT, AU. AZ. BA, BB, BG BR. 
BY CA. CH. CN. CR. CU. CZ. DE. DK. DM. EE, ES. FX, 
GB* GD. GE, GH. GM, HU, ID. IL. IS, JP. KE, KG, KP. 
KR* KZ LC. LK. LR, LS. LT. LU. LV, MA, MD, MG. 
MK MN, MW. MX. NO, NZ, PL, PT. RO. RU. SD. SE. 
SG 'Sl. SK, SL, TJ. TM, TR, TT, TZ. UA. UG, US, UZ, 
VN. YU. ZA. ZW. ARIPO patent (GH, GM, KE. LS. MW. 
SD,* SL, SZ. TZ, UG. ZW). Eurasian patent (AM. AZ. BY. 
KG KZ. MD. RU. TJ. TM). European patent (AT, BE. CH, 
CY; DE, DK. ES. PI. FR, GB. GR. IE. IT. LU. MC. NL. 
PT SE). OAPI patent (BF. BJ. CF, CG. CI, CM. GA. GN, 
GW. ML, MR. NE. SN. TD. TG). 



Published 

With international search report. 

(SS) Date of publication of the international search report: 

L.a« o p ^ ^^^^^^^ ^^^^ (05. 10.00) 



; (54) ritie: IN UTERO TRANSPLANTATION OF HUMAN MESENCHYMAL STEM CELLS 
(57) Abstract 

' transplantation. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT. 



AL 

AM 

AT 

AU 

AZ 

BA 

BB 

BE 

BF 

BG. 

BJ 

BR 

BY 

CA 

CF 

CG 

CH 

a 

CM 
CN 

cu 
cz 

DE 
DK 
EE 



Albania 

Arrnenia 

Austria 

Australia 

Azerbaijan 

Bosnia and Herzegovina 

Barbados 

Belgium 

Burkina Faso 

Bulgaria 

Benin 

Brazil 

Belarus 

Canada 

Central African Re{)ublic 

Congo 

Switzerland 

C6te d'lvoirc 

Cameroon 

China 

Cuba 

Czech Republic 
Gennany 
Denmark 
Estonia * 



£S 


Spain 


LS 


Lesotho 


SI 


FI 


Finland 


LT 


Lithuania 


SK 


FR 


France ■ 


LU 


Luxembourg 


SN 


GA 


Gabon 


LV 


Latvia 


sz 


GB 


United Kingdom 


MC 


Monaco 


TD 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


GH 


Ghana 


MG 


. Madagascar 


TJ 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


GR 


Greece 




Republic of Macedonia 


TR 


HU 


Hungary 


ML 


Mali 


TT 


IE 


Ireland 


MN 


Mongolia 


UA 


IL 


Israel 


MR 


Mauritania 


UG 


IS 


Iceland 


MW 


Malawi 


US 


IT 


Italy 


MX 


Mexico 


uz 


JP 


Japan 


NE 


Niger 


VN 


KE 


Kenya 


NL 


Netherlands 


YU 


KG 


Kyrgyzstan 


NO 


Norway 


zw 


KP 


Democratic People's 


NZ 


New Zealand 






Republic of Korea 


PL 


Poland 




KR 


Republic of Korea 


PT 


Portugal 




KZ 


Kazakstan 


RO 


Romania 




LC 


Saint Lucia 


RU 


Russian Federation 




LI 


Liechtenstein 


SD 


Sudan 




LK 


Sri Lanka 


SE 


Sweden 




LR 


Liberia 


SG 


Singapore 





Slovenia 

Slovakia 

Senegal 

Swaziland 

Chad 

Togo 

Tajikistan 

"nukraenisian 

Turkey 

Trinidad and Tobago 

Ukraine 

Uganda 

United Slates of America 

Uzbekistan 

Vict Nam 

Yugoslavia 

Zimbabwe 



BNSDOCID: <WO 0029002A3_I_> 



INTEilNATIONAL SEARCH REPORT 



tntc onal Application No 

PCT/US 99/26927 



A;«GT^SSIfiGATI(3N OF SUBJECT MATTER , , ,_^/^ , 

■p|- 7 A61K35/12 A61K35/28 A61K48/00 AG1K67/027 

V ci2iii:5/oo 

According to International Patent gassification (IPC) of to both national dassification and IPC 



C12N5/00 



B. RELOS SEARCHED 



Minimum documentation searched (classification system followed by ctasstfication symtxais) 

PC 7 A61K AOIK C12N 



Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched 



Qectronic data base consulted <hjring the intemationai search (name of data base and, where practical, search terms used) 



a DOCUMENTS CONSIDERED TO BE RELEVANT 



Category • Citation of document, with incScation. wfiere appropriate, of the retevantt passages 



Relevant to daim No. 



SROUR E F ET AL.: "Sustained human 
hematopoiesi s in sheep transplanted in 
utero during early gestation with 
fractionated adult human bone marrow 
cells" 
BLOOD, 

vol. 79, no. 6, 

15 March 1992 (1992-03-15), pages 
1404-1412, XP000569644 
the whole document 



1-8 



Further documertts are listed in the continuabon of box C- 



Patent family members are Osted in anr^ex. 



' Special categories of cited documents : 

"A" document defining the general state of the art wfiich is not 

considered to be of particular relevance 
'E' earlier document but published on or after the intemationai 

fiiir^ date 

"L" document wfiich may throw doubts on priority claim(s) or 
wfvch is dted to establish the putstication date of another 
dtation or other special reason (as spedfied) 

•O' document i nl e i ili i g to an oraf disctosure. use, exhtoibon or 
other means 

•P* document putiTished prior to the intemationai filing date txjt 
later than the priority date daimed 



"T" later document published after the intemationai ffling date 
or priority date and not tn conflict with the application but 
cited to understand the principle or theory undertying the 
invention 

"X" document of particular relevance; the claimed invention 
cannot be considered novel or cannot be considered to 
invdve an inventive step when the document is taken alone 

"V document of particular relevance; the claimed Invention 

cannot t)e considered to involve an inventive step when the 
document is combined with one or more other such docu- 
ments^ such combination being obvious to a person skiBed 
in the art 

document member o( tf>e same patent family 



Date of the actual comptetion of the intemationai search 



9 June 2000 



Date of mailing of the intemationai search report 



27/06/2000 



Name arKl mafiing address of the ISA 

European Patent (Office. P.B. 5818 Paterttlaan 2 
NL-2280HVRi^wijk 
Tel. (+31 -70) 340-2040. Tx. 31 651 epo ni. 
Fax: (+31-70) 340-3016 



AutfKKtzed officer 



Teyssier, B 



Form PCT/1SA/210 (second 6ho«t) (July 1902) 



page 1 of 3 



intei^Pt nal search report IB- 

W. TTra* ..iBl Application No 

I PCT/US 99/26927 

C.(Cofitlnuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT 



Category * 


Citation ot ckx^ment with inclicalion.whefB appropnate, ot the relevant paseagee 


Relevant to daim ^k>. 


X 
A 


PROCKOP D J: "Marrow stromal cells as 
stem cells for nonhematopoletic tissues" 
SCIENCE, 

vol. 276, no. 5309, 

4 April 1997 (1997-04-04), pages 71-74, 
XP002139788 

pages 73-74, "Potential uses in cell and 
gene therapy" 


1-3 

4-8. 
24-27 


X 
A 


PEREIRA R F ET AL.: "Cultured adherent 
cells from marrow can serve as 
long-lasting precursor cells for bone, 
cartilage, and lung in irradiated mice" 
PROC. NAT'L. ACAD. SCI. USA, 
vol. 92. May 1995 (1995-05), pages 
4857-4861. XP002139789 
cited in the application 
pages 4859-4861, "Discussion" 


1-3 

4-8, 
24-27 


A 


WO 97 10348 A (UNIV CALIFORNIA) 
20 March 1997 (1997-03-20) 
page 3, line 31 -page 5, line 14 
page 8, line 21 -page 11, line 27 


1-8, 
24-27 


A 


WO 94 26884 A (BIOTECHNOLOGY RES & DEV) 

24 November 1994 (1994-11-24) 

page 1 -page 4, line 26 

page 7, line 8 - line 27; claims 1,35 


9-23 


P,X 


LIECHTY K W ET AL.: "Distribution and 
engraftment of human mesenchymal stem 
cells (HSC) after in utero transplantation 
in fetal sheep." 
BLOOD, 

vol . 92, no. 10 Suppl .1, 

15 November 1998 (1998-11-15), page 117A 

XP000914604 

40th annual meeting of the American 
Society of Hematology, 4-8/12/1998 
abstract 474 


1-27 


P.X 


WO 99 46366 A (OSIRIS THERAPEUTICS INC) 

16 September 1999 (1999-09-16) 

page 1 -page 2, line 21 

page 11, line 15 -page 13, line 28 

-/— 


1-3 



Form PCT/ISA^IO (oontinuaiiGn at second shoot) (Juty 1092) 

page 2 of 3 

BNSDOCID: <WO O029O02A3_l_> 



INTERNATIONAL SEARCH REPORT 



• C.(Contlnuatlon) OOCUMEKTS CONSIDERED TO BE RELEVAMT 

Otation of document, with indicatian.where appropriateTot the relovant passages 

ALMEIDA-PORADA G ET AL: "Human bone 
marrow stromal cell progenitors are able 
to induce donor specific tolerance after 
in utero transplantation." 
BLOOD , 

vol. 94, no. 10 SUPPL. 1 PART 1, 
15 November 1999 (1999-11-15), page 40a 
XP002139877 

41th annual meeting of the American 
Society of Hematology, 3-7/12-1999 
abastract 167 

DEVINE S ET AL.: "Studies of mesenchymal 
stem cells in non-human primates: 
Evaluation of toxicity and engraftment" 
BLOOD, 

vol . 94, no. 10 Suppl . 1, 
15 November 1999 (1999-11-15), page 391a 
XP002139790 

41th annual meeting of the American 
Society of Hematology, 3-7/12/1999 
abstract 1733 



Inte onal Apptlcatkm No 

PCT/US 99/26927 



Relevant to daim No. 



Form PCTASAfilO (oontinuation sooond sh^t) (Jiiy 1992) 



page 3 of 3 



INTE^^" INAL SEARCH REPORT 

•ntormotlon on patent taihlly rnembaw 



tntei jnal Application No 

PCT/US 99/26927 



Patent document 


Publication 




Patent family 




Publication 


Cftod in sodrch report 


a die 




membeits) 




date 


WO 9710348 A 



20-03-1997 


AU 


7238796 


A 


01-04-1007 


WO 9426884 A 


24-11-1994 


US 


5523226 


A 








AT 


184647 


T 
1 


1 1 n— 1 GOO 






AU 


694126 


R 

EJ 


xU u/— Xyyo 






AU 


7019394 


A 


12-12^1994 






DE 


69420726 


0 


21-10-1999 






DE 


69420726 


T 


27-04-2000 






EP 


0701608 


A 


20-03-1996 






ES 


2140542 


T 


01-03-2000 






US 


5942435 


A 


24-08-1999 


WO 9946366 A 


16-09-1999 


AU 


2904299 


A 


27-09-1999 



Form PCT/lSA/210 (patent tamfly annex) (JUy 1992) 

BNSDOCID: <WO 0029002A3J_>