AU2005202789B2 - Differentiated human embryoid cells and a method for producing them - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM TECHNION RESEARCH AND DEVELOPMENT FOUNDATION LTD.

Invention Title: DIFFERENTIATED HUMAN EMBRYOID CELLS AND A METHOD FOR PRODUCING THEM The following statement is a full description of this invention, including the best method of performing it known to me/us: WO 00/70021 -la PCT/ILOO/00270 DIFFERENTIATED HUMAN EMBRYOID

CELLS

AND A METHOD FOR PRODUCING

THEM

FIELD OF THE INVENTION The present invention is in the field of cell biology and more specifically relates to methods of in vitro differentiation.

BACKGROUND OF THE INVENTION Embryonic stem (ES) cells are derived from totipotent cells in an embyro.

Murine ES cells have been shown to be pluripotent cells capable in vitro of terminal differentiation into cells of the mesoderm, ectoderm and endoderm lineages (Robertson, 1987; Dushnik-Levinson and Benvenisty, 1995). The pluripotency of murine embryonic stem cells has been established on the basis of three criteria: When undifferentiated murine ES cells are injected into the cavity of a blastocyst and the blastocysts are subsequently implanted into pseudo-pregnant mice, chimeric mice develop. The injected ES cells contribute to all cell types, including the germ layer. Thus, in the next generation, mice with the genotype of the ES cells are born (Capecchi, 1989; Rossant and Joyner, 1989).

When murine ES cells are injected subcutaneously into syngeneic mice, teratoma tumors develop. These tumors comprise cells of all three embryonic origins (endoderm, ectoderm and mesoderm) (Wobus et al., 1984).

2 When murine ES cells are allowed to aggregate in C vitro so as to form embryoid bodies(EBs), the Scells differentiate in the EBs into various cell types (Robertson. 1987).

c- During maturation of murine ES cells in vitro, in 00 addition to morphological changes, a cell may acquire a (C molecular marker characteristic of a differentiated cell C-q type, such as k-globin (a marker of hematopoietic cells) 0 10 (Wiles and Keller, 1991; Lindenbaum and Grosveld, 1990), C-i neurofilament-68kd protein (a marker of neuronal cells) (Bain et al., 1995; Levinson-Dushnik and Benvenisty, 1997), and albumin (a marker of hepatic cells) (Levinson- Dushnik and Benvenisty, 1997).

ES cell lines have also been established from primates such as the common marmoset (Thomson, 1996)), and the rhesus monkey (Thomson, 1995)). However, EB formation by marmoset ES cells is inconsistent and asynchronous, and differentiation of the rhesus ES cells is disorganized and vesicular structures do not form (Thomson, 1998a).

Human ES cell lines have been established derived from human embryos produced by in vitro fertilization (Thomson, 1998b). The embryos were cultured to the blastocyst stage, and inner cell masses comprising ES cells were isolated and cultured. These human ES cells are only known to be capable of differentiating when in teratomas (Thomson, 1998b). When cultured in vitro, the human ES cells have normal karotypes, express telomerase activity, and proliferate. However, the inability to produce EBs from non-human primate ES cells lead to the H:\terryr\Keep\Speci\P57369 DIV Speci Claims Yissum Research June 2005.doc 27/06/05 2a 0 belief that EBs could also not be formed from human ES C-q cells (Thomson, 1998a).

In the claims of this application and in the description CI 5 of- the invention, except where the context requires otherwise due to express language or necessary 00 implication, the words "comprise" or variations such as Cq "comprises" or "comprising" are used in an inclusive C-i sense, i.e. to specify the presence of the stated features S 10 but not to preclude the presence or addition of further C-I features in various embodiments of the invention.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

SUMMARY OF THE INVENTION The present invention is based on the finding that, contrary to the aforementioned belief that human ES cells do not form EBs, under appropriate conditions, human embryoid bodies (hEBs) may be obtained in vitro, from human ES (hES) cells suspended in a liquid medium. These hEBs contain mesoderm, ectoderm and endoderm cell lineages and may be used as a source of cells of the different lineages, e.g. for transplantation or inoculation into human recipients. By H:\terryr\Keep\Speci\P57369 DIV Speci Claims Yissum Research June 2005.doc 27/06/05 WO 00/70021 PCT/IL00/00270 0 incubating the hEBs. these basic cell lineages can differentiate into a wide variety c1 of different cell types. e.g. cells having characteristics of cardiac cells, neural stem Scells, and others.

The hEBs in accordance with the invention mav be used as a source of cells for use in transplantation or inoculation into a human recipient, in order to treat Svarious diseases.or disorders, to assist in tissue repair, to substitute for degenerated 00 tissue.

Thus. in accordance with one aspect, the present invention provides a 0human embryoid body (hEB).

1 io The hEB, in accordance with the invention, is preferably obtained by in vitro culturing of hES cells in a vessel under conditions in which the cells or aggregates thereof do not adhere to the vessel walls.

In accordance with another aspect, the invention provides a process for obtaining at least one human-derived embryoid body (hEB), comprising: providing human embryonic stem (hES) cells; growing the hES cells in vitro in a vessel under conditions in which said cells undergo differentiation and the cells or aggregates thereof do not adhere to the vessel wall; and incubating for a time sufficient to develop hEBs from said cells.

The conditions whereby the hES cells do not adhere to the vessel wall include culturing the cells in a vessel having walls made of a material to which the cells or aggregates thereof are incapable of adhering, adding one or more factors into the medium which prevent adherence of the cells to the vessel walls etc.

Conditions whereby the hES undergo differentiation include the absence of inhibitors of differentiation such as leukemia inhibitory factor and fibroblast growth factor.

The invention also provides a process for preparing cells of a defined cell lineage comprising: providing human embryonic stem (hES) cells; WO 00/70021 -4- PCT/IL00/00270 0 growing the hES cells in vitro in a vessel under conditions under cI which the cells undergo differentiation and said cells or aggregates thereof do not Sadhere to the vessel wall: incubating for a time sufficient to obtain at least one embryoid body (EB) containing cells of said defined cell lineage; and isolating said cells from said EB.

oo In accordance with one embodiment, the cells of the defined cell lineage may be inoculated or transplanted into a human recipient to treat a certain human disease or condition, to allow tissue or organ repair, etc. Thus, in accordance with N to1 this preferred embodiment, the cells of the defined cell lineage are formulated in a manner to allow their inoculation or transplantation into the human recipient.

The present invention also provides, by another of its aspects, an inoculable or transplantable preparation comprising cells of said defined cell lineage together with a physiologically acceptable carrier which is compatible with said cells. The term "compatible with said cells" should be understood as a medium which ensures the viability of said cells until they are inoculated or transplanted into the human recipient.

The cells of the defined cell lineage may be induced to undergo furthr differentiation and transplanted or inoculated into the individual as a cell suspension or alternatively, they may be cultivated to form a cell mass or an in vitro tissue, and the mass or the tissue then being transplanted into the individual.

The hES cells from which the hEBs of the invention are derived may be obtained from established lines see Thomson et al., 1998b, supra) or may be prepared de novo from human embryos which were produced by in vitro fertilization.

BRIEF DESCRIPTION OF THE DRAWINGS In order to understand the invention and to see how it may be carried out in practice, a preferred embodiment will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: WO 00/70021 PCT/IL00/00270 Fig. lA shows cystic hEBs from 3 to 14 days after transfer to petri dishes produced in accordance with the invention. I: simple EB; II III: cavitated EB; IV: Scystic hEB. Scale bar. 40 pm.

t"l Fig. 1B shows 5 LLm paraffin embedded sections of hEBs strained with hematoxylin and eosin I,II and IV) or DAPI (III) I: scale bar 80 prn; II& III 00 scale bar 20 pm; IV: scale bar 4 pmn.

Fig. 2 shows the expression of cell specific genes in hES cells and cystic hEBs; 0Fig. 3 shows in situ hybridization analysis of expression of a-fetoprotein io (cFP), -globin -Glob), neurofilament 68 kd protein (NF) and a-cardiac actin (cAct) in 20 day old Ebs (Con indicates control); Fig. 4A shows a pulsating hEB in a relaxed state. Scale bar, 40 pmn; Fig. 4B shows the pulsating hEB of Fig. 4A in a subsequent contracted state. Scale bar, 40 pm; and Fig. 4C shows serial sections from a pulsating hEB including a section stained with H&E, a hybridized section showing a-cardiac actin expression (cActin), and a control section hybridized with non-specific RNA (Control). Scale bar,100 inm.

MATERIALS AND METHODS Formation of human cystic EBs Human ES cells (H9 clone 10) were grown on mouse embryo fibroblasts in a culture medium consisting of 80% KnockOutTM DMEM (an optimized Dulbeco's modified Eagle's medium for ES cells, Gibco-BRL), 20% KnockOut T M SR (a serum-free formulation, Gibco-BRL), 1 mM glutamine (Gibco-BRL), 0.1 mM P-mercaptoetanol (Sigma). 1% non-essential amino acids stock (Gibco-BRL), 3 units/ml leukemia inhibitor factor (LIF) (Gibco-BRL) and 4 ng/ml basic fibroblast growth factor (bFGF) (Gibco-BRL). Under these conditions most of the cells are kept in an undifferentiated state. To induce formation of hEBs, the ES cells were transferred to plastic petri dishes to prevent their adherence to the dish and WO 00/70021 PCT/IL00/00270 0 promote their aggregation. The concentration of the cells was about 10' cells per ml. The hEBs were then cultured in the above culture medium not containing Sleukemia inhibitor factor or basic fibroblast growth factor.

Detection of expression of cell specific genes in cystic hEBs.

Total RNA was extracted from cells as previously described (Chirgwin S et al., 1979) and cDNA was synthesized from 1 gg of total RNA, using a random ClN hexamer (pd(N) 6 as primer (Pharmacia Biotech) and M-LMV Reverse STranscriptase (Gibco-BRL). cDNA samples were subject to polymerase chain N 1to reaction (PCR) amplification with specific DNA primers. PCR was performed under linear conditions in order to reflect the original amount of the specific transcript. The PCR primers used and the reaction conditions were: a-fetoprotein: AGAACCTGTCACAAGCTGTG and GACAGCAAGCTGAGGATGTC- Product: 676 base pairs 20 cycles at 600C in 1 mM MgCl2; -globin: GACTGAGAGGACCATCATTG and TCAGGACAGAGGATACGACC Product: 397 bp. 25 cycles at 600C in 1 mM MgCl 2

GAPDH;

AGCCACATCGCTCAGACACCA and GTACTCAGCGGCCAGCATCG Product: 302 bp. 20 cycles at 600C in 1 mM MgCl 2 PCR products were analyzed by Western Blot hybridization (Southern, 1975). Probes were radiolabeled by random priming (Boehringer Mannheim) using [a-3P]dCTP (3000 ci/mM, NEN Life Science Products).

In situ hybridization analysis of hEBs EBs were stained with hematoxylin and eosin or hybridized to specific RNA probes labeled with a fluorescent reagent (Grifnfan et al., 1998b) and pm paraffin embedded serial sections were obtained. Control hybridizations were performed with non-specific RNA. The probes used were 50-mer 2'-O-methyl biotinylated cRNA of either a-fetoprotein

TTGTCCCTCTTCAGCAAAGC

AGACTTCCTGTTCCTGGCCTTGGCAGCATT, -globin TGATGGTCCTCT o3 CAGTCTTGGTCAGAGACATGGCGGCAGGGTGGGCAGCT, Neurofilament WO 00/70021 -7- PCT/IL00/00270 O 68 kd protein CCTGCGTGCGGATGGACTTGAGGTCGTTGCTGATGGCG GCTACCTGGCTC. or a-cardiac actin CGGTGGACAATGGATGGGCCTG

CCTCATCGTACTCTTGCTTGCTAATCCA.

U--

EXAMPLES

Example 1: Formation of cystic hEBs Fig. 1A shows several hEBs 3 to 14 days after having been transferred to Ci plastic petri dishes and cultured in medium not containing leukemia inhibitor factor O or basic fibroblast growth factor. Initially, simple hEBs form (Fig. 1A-I).

S 10 Subsequently, the center of the bodies became cavitated (Fig. 1A, II and III), and the bodies began to accumulate fluid, turning to cystic hEBs (Fig. 1A, IV). 20 days after initiation of cellular aggregation most of the structures were cystic and they included a variety of epithelial and mesenchymal cells. Fig. 1B shows 5 Jim paraffin embedded sections of 20 day old hEBs stained with H&E (I-IV) or DAPI DAPI staining of the nuclei revealed in some of the cells condensed chromatin that probably corresponds to the apoptosis occurring in the center of the hEB (Fig. 1B-III).

Example 2: Expression of cell specific genes in cystic hEBs.

In order to examine the differentiation status of the EBs, RNA was extracted from hES cells grown on mouse embryo fibroblasts, and from 20 day old EBs.

cDNA was synthesized using these RNA samples and expression analysis of several genes was performed by RT-PCR with various human specific DNA primers. The RT-PCR analysis was performed under non-saturating linear conditions. The identity of the amplified DNA product in the PCR assay was verified by sequence analysis. The lane marked EB in Fig. 2 shows that the hEBs express a-fetoprotein (aFP), an endodermal marker (Krumlauf et al., 1985), and 4-globin, a marker of hematpoietic cells (Leder et al., 1985)). Very low levels of these markers were observed in the hES cells (Lane ES). The house keeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), served as an internal control. Linearity of the observed signal is demonstrated by the "1/10 column" WO 00/70021 -8- PCT/IL00/00270 0 showing a PCR assay performed with one-tenth the amount of cDNA used in the

O

iN PCR assay of the hEB sample.

r' Example 3: In situ hybridization analysis of hEBs To regionally characterize the differentiating cells within the EBs by in situ 0 hybridization. expression of four cell-specific molecular markers, all of which oo transcribe very early during embryonic differentiation, was examined. Serial sections of the hEBs were hybridized with RNA probes specific to c-fetoprotein S(caFP) (Krumlauf et al., 1985), -globin (4-glob) ((Leder 1985), a-cardiac actin c l to (cAct) (Sassoon et al., 1988)) or neurofilament 68kd protein (NF) (Julien et al., 1986)). As shown in Fig. 3, each of these molecular probes specifically labels distinct regions in the EBs. This suggests that the labeled cells in each section were either clonal (derived from a common progenitor cell), or were affected by the same signals and differentiated into the same specific lineage.

Example 4: Characterization of a pulsating hEB Differentiation of murine ES cells in EBs into the myocardial lineage has been shown to produce pulsating muscle (Sanchez et al., 1991). The existence of cardiac muscle cells in hEBs was demonstrated by in situ hybridization of sections from a human EB with a-cardiac actin, a marker of embryonic myocardial cells (Sassoon et al., 1988). Figs. 4A and B shows a large vacuated hEB including cardiac muscle cells that were pulsating at a rate of about 30 beats/min. The hEB was first photographed in a relaxed state (Fig. 4A) and then in a subsequent a contracted state (Fig. 4B). Fig. 4C shows serial sections of a cell including a section stained with hematoxylin and eosin a hybridized section showing a-cardiac actin expression (cActin), and a section hybridized with non-specific RNA (Control).

WO 00/70021 9- PCTJILOO/00270 References Bain. Kitchens. Yao, Huettner. Gottlieb, Developmental ;Z Biology. 168:342-57 (1995).

c-I Capecchi. Science. 244:1288-92 (1989).

Chirgwvin. Przybyla. Macdonald. Rutter, Biochemistry, 00 18:5294-9 (1979).

Dushnik-Levinson, Benvenisty, Biology. of the Neonate. 67:77-83, (1995).

Julien, Meyer, Flavell, Hurst. Grosveld, Brain Research, 387:243-50 c-i (1986).

Krumlauf. Hammer, Tilghman, Brinster, Molecular and Cellular Biologv, 5:1639-48 (1985).

Leder, Weir, Leder, Molecular and Cellar Biology, 5:1025-33 (1985).

Levinson-Dushnik, Benvenisty, Molecular and Cellular Biology, 17:38 17-22 (1997).

Lindenbaum, Grosveld, Genes and Development, 4:2075-85 (1990).

Robertson, in Teratocarcinoma and Embryonic Stem Cells: A Practical Approach, R. EJ, Eds. (IRL Press, Oxford, 1987) pp. 71-:112.

Rossant A.L. Joyner, Trends-in Genetics, 5:277-83 (1989).

Sanchez, Jones, Gulick, Doetschrnan, Robbins, Journal of Biological Chemistry, 266:22419-26 (199 1).

Sassoon, Gamner, Buckingham, Development, 104:155-64 (1988).

Southern, Journal of Molecular Biology, 98:503-17 (1975).

Thomson, et al., Biology ofReproduction, 55:254-9 (1996).

Thomson, JA.. et al., Proceedings of the National Academy of Sciences of The United States ofAmerica, 92:7844-8 (1995).

Thomson, J.A. and Marshall, VS., Curr. Top. Dev. Biol. 38. 13 3 (1988a) Thomson, et al., Science, 282:1145-7 (1998b).

WO 00/7002 1 10- PCT/ILOO/00270 Wiles. Keller. Developm7ent. 111:259-67 (1991).

Wobus. Holzhausen. Jdikel. Sch~neich, E-Cperimiental Cell Research. 152:212-9 (1984).

00

Claims (13)

1. An isolated human embryoid body (hEB) derived from human embryonic stem cells and containing mesoderm, C 5 ectoderm and endoderm cell lineages. 00

2. An hEB according to claim 1, obtained by in vitro C-q culturing of human embryonic stem (hES) cells in a vessel C-q under conditions in which the cells undergo 0 10 differentiation and the cells or aggregates thereof do not Ci attach to the vessel walls.

3. An hEB according to claim 1 or claim 2, comprising cells displaying characteristics of cardiac cells.

4. An hEB according to any one of claims 1 to 3, comprising cells expressing a marker selected from the group consisting of a-fetaprotein, -globin, a-cardiac actin and neurofilament 68 kd.

A process for obtaining at least one human-derived embryoid body (hEB), comprising: providing isolated human embryonic stem (hES) cells; growing the hES cells in vitro in a liquid growth medium in a vessel under conditions under which said cells undergo differentiation and said cells or aggregates thereof do not adhere to the vessel wall; and incubating for a time sufficient to develop hEBs from said cells.

6. A process according to claim 5, wherein said conditions comprises the absence of leukemia inhibitory H:\terryr\Keep\Speci\P57369 DIV Speci Claims Yissum Research June 2005.doc 27/06/05 12 0 factor and fibroblast growth factor from the growth C-i medium.

7. A process according to claim 5, wherein said C 5 condition comprises culturing cells in a vessel having walls of a kind to which cells do not adhere. 00 Cq

8. A process for preparing cells of a defined cell eCq lineage, comprising: providing isolated human embryonic stem (hES) cells; C growing the hES cells in vitro in a vessel under conditions in which said cells undergo differentiation and said cells or aggregates thereof do not adhere to the vessel wall; incubating for a time sufficient to obtain at least one human embryoid body (hEB)containing cells of said defined cell lineage; and isolating said cells from said hEB.

9. A process according to claim 8, wherein said cells are formulated for inoculation or implantation into a recipient individual.

An injectable or implantable preparation, comprising cells obtained by the process of claim 8 or claim 9 together with a physiologically acceptable carrier compatible with said cells.

11. An implantable cell mass or tissue obtained by a process according to claim 8 or claim 9.

12. Cells produced according to a process according to claim 9. H:\terryr\Keep\Speci\P57369 DIV Speci Claims Yissum Research June 2005.doc 27/06/05 13 CM

13. An isolated hEB according to claim 1 substantially as hereinbefore described with reference to any one of the examples. CM Dated this 2 7 th day of June 2005 00 YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW C UNIVERSITY OF JERUSALEM CM and O 10 TECHNION RESEARCH AND DEVELOPMENT FOUNDATION LTD CM By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\terryr\Keep\Speci\P57369 DIV Speci Claims Yissum Research June 2005.doc 27/06/05