AU757036B2

AU757036B2 - Derivation of cells and tissues from embryonic pre-stem cells for transplantation therapies

Info

Publication number: AU757036B2
Application number: AU28799/99A
Authority: AU
Inventors: Gary D Hodgen
Original assignee: Medical College of Hampton Roads
Current assignee: Medical College of Hampton Roads
Priority date: 1998-02-27
Filing date: 1999-02-26
Publication date: 2003-01-30
Anticipated expiration: 2019-02-26
Also published as: EP1056835A4; JP2002504362A; WO1999043785A1; CA2320423A1; EP1056835A1; AU2879999A

Description

WO 99/43785 PCT/US99/04188 DERIVATION OP CELLS AND TISSUES FROM EMBRYONIC PRE-STEM CELLS FOR TRANSPLANTATION THERAPIES Background of the Invention The present invention relates to the derivation of cells and tissues from embryonic pre-stem cells for transplantation therapies.

Summary of the Invention This invention relates to the use of dispersed morula cells in preference to inner cell mass (ICM) from blastocysts. The morula stage is the last pre-embryonic stage without expression of any differentiation, making these cells (pre-stem cells) all progenitors of embryonic stem cells (ESCs) later present in blastocysts.

Conversely, the ICM from the blastocyst is already differentiated from trophoblastic cells, which are by then destined to become part of the placenta.

This invention also relates to the use of chimeric introductions into pre-stem cell cultures and stem cell propagations in culture. That is, "teacher-cells" or spent media from them, that derived from other sources adults, cord blood, fetal tissues, etc.) will "teach" undifferentiated pre-stem cells how to convert to our sought-after therapeutic cell population both more rapidly and more preferentially.

This invention also relates to the identification and use of certain early markers of stem cell SUBSTITUTE SHEET (RULE 26) 2 differentiation, such as Fe++ sequestration, hemoglobin accumulation, myeloid fibers, insulin synthesis, dopamine loading, etc.

Other features and advantages of the present invention will become apparent from the following description of the invention.

Detailed Description of the Invention The present invention provides for a method of isolating and propagating a line of embryonic stem cells that originates from either morulae (pre-stem) or blastocyst (ICM stem cells). Therefore, Morula stage, undifferentiated pre-stem cells will be used as progenitors of stem cell populations. The propagated line of embryonic stem cells are then used for the purpose of transplanting 15 cells, tissues or organs.

Accordingly, in a first aspect of the present invention there is provided a method of e isolating and propagating a line of pluripotent embryonic stem cells that originates from either morulae or blastocyst comprising: propagating the stem cells in a culture medium; and adding apoptotic factors to the culture medium in order to achieve clonal properties of the propagated stem cells by eliminating contaminating members of the stem cells that did not properly differentiate.

2a The propagation of stem cells can be initiated by formation of chimeric inner cell mass cells. Chimeric ICMs will be developed from blastocysts. From such ICMs, superior stem cell cultures are derived. Preferably, the formation of chimeric inner cell mass cells comprises nuclear transplantation, mitochondrial substitution, or cytoplasmic depletion.

Preferably, at least one regulatory factor is used to propagate the line of embryonic stem cells. More preferably, the-regulatory factor is derived from "Teacher cells" or "Teacher cells'" spent culture medium. "Teacher cells" will be introduced into less differentiated pre-stem or early stage stem cells to accelerate propagation of target stem cells. Alternatively, spent media from .15 *o H:\MraR\Keep\Speci\P391 76 .dcIc 30/10/02 WO 99/43785 PCT/US99/04188 3 "teacher cells" can be used to accelerate the propagation of the target stem cells.

In a preferred embodiment, the embryonic stem cells are cultured in a medium in the presence of at least one agent or cytokine in order to differentiate into target specific cells or tissues. Preferably, the agent or cytokine is selected from the group consisting of IL-1, TNF-a, IL-6, PTH, PDGF, PGE,, cAMP, estrogens, antiestrogens, progestins, anti-progestins, cortisol, GH, androgens, 1 3

/T

3 VGEF and cyclosporin. Also preferably, the concentration of the agent or cytokine in culture medium is from about 1.0 pg/ml to about 10.0 ng/ml.

In another preferred embodiment, the target specific cells are selected from the group consisting of nerve cells, bone cells, immune cells, and pancreatic beta cells.

Techniques and parameters for the use of a broad spectrum of early stage metabolic markers are developed.

Some such markers are, for example: Fe++ sequestration, Hg accumulation, myeloid fibers, nerve growth factor, apoptotic factors, insulin synthesis, dopamine loading, hemoglobin loading, etc. Additionally, other early markers of embryonic stem cells can be identified.

Specific techniques are developed to demonstrate the foregoing. In one embodiment of the invention, embryonic stem (ES) cells are derived from either morula or blastocyst stage embryos by placing cells on fibroblast feeder layers. The colonies are evaluated for differentiation state using accepted markers. Further SUBSTITUTE SHEET (RULE 26) 4 evaluation is done by injecting ES cells into surrogate embryos to produce chimeras and evaluating the contribution of the ES cells to the adult tissues. Finally, ideal colonies of ES cells are used as nuclear donors for nuclear transplantation.

Clonal properties of the propagated stem cells are achieved by adding apoptotic factors to the culture medium to eliminate contaminating members of the stem cells that did not properly differentiate.

10 Preferably, agents are selected from the group consisting of IL-1, TNF--a, IL-6, PATH, PDGF, PGE 2 cAMP, estrogens, anti-estrogens, progestins, antiprogestins, cortisol, GH, androgens, I3/T 3 VGEF and cyclosporin.

15 Alternatively, the propagation of the line of embryonic stem cells is done in vivo by transplanting "Teacher cells" into an area sufficiently close to the embryonic stem cells to allow for at least one regulatory factor made by the teacher cells to contact the embryonic 20 cells.

Accordingly in a second aspect of the present invention there is provided a method of propagating a line of pluripotent embryonic stem cells in vivo that originates from either morulae or blastocyst comprising transplanting teacher cells into an area sufficiently close to the embryonic stem cells to allow for at least one agent or cytokine made by the teacher cells to contact the embryonic cells.

4a The presence or absence of different concentrations of calcium can be used to regulate the propagation of the line of embryonic stem cells.

Preferably, the propagated line of embryonic stem cells is grown in a three dimensional manner before being used for transplantation.

This invention will allow for the efficient, safe and commercially viable derivation of cells and tissues H:\MaraP\Keep\Speci\P39176.dcc 30/10/02 5 from embryonic pre-stem cells for transplantation therapies. Specifically, growing-out of human blastocysts at a rate greater than 50% from the 2-cell stage of the pre-embryo should be achieved. Also, efficient harvesting of either morula stage pre-stem cells and/or stem cells isolated from the inner cell mass of blastocysts should be achieved. These embryonic pre-stem and stem cell populations should preferably remain viable in culture for more than one week.

Throughout this specification and the claims, the words "comprise", "comprises" and o ."comprising" are used in a non-exclusive sense, except where the context requires otherwise.

It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

20 0 Example 1 Clonal production of stem cells will be undertaken. These clones will respond to the ambient levels of glucose in their milieu, and in turn, insulindependent diabetes would be treated by transplanting these -stem cell lines to served by a peripheral blood supply.

the insulin secretory cells must accomplish renewal y propagation through mitogenic proliferation.

5a Example 2 Pluripotent stem cells will be isolated and directed to differentiate into hemopoietic destinies.

Therefore, tissues derived from the blood cell group or beta cells of the immune response system will be replaced in deficient patients suffering from conditions such as HIV infection, post-chemotherapy, or irradiation depletion.

Culture condition in vitro will direct the rate and degree of differentiation manifested by these pluripotent stem cells, such as The presence of "teacher cells" or certain additives to the media, e.g. cytokines.

Example 3 e H:\MaraR\Keep\Speci\P39176.dOC 30/10/02 WO 99/43785 PCT/US99/04188 6 The inherent capabilities of stem cells will be modified by formation of chimeric cell lines that incorporate "hybrid" metabolic functions that when transplanted will provide the transplant recipient with long-term relief from organ/tissue deficiencies. For instance, the production of dopamine in situ can modify neurological treatments for patients manifesting muscular rigidity and loss of motor control in disease states such as Parkinson's disease. Unlike pharmaceutical therapeutics which are partially effective temporarily, transplantation of chimeric stem cells that regulate the production dopamine and serotonergic factors will offer these patients superior outcomes.

The invention has been described in terms of preferred embodiments thereof, but is more broadly applicable as will be understood by those skilled in the art. the scope of the invention is therefore limited only by the following claims.

SUBSTITUTE SHEET (RULE 26)

Claims

1. A method of isolating and propagating a line of pluripotent embryonic stem cells that originates from either morulae or blastocyst comprising: propagating the stem cells in a culture medium; and adding apoptotic factors to the culture medium in order to achieve clonal properties of the propagated stem cells by eliminating contaminating members of the stem cells that did not properly differentiate.

2. The method of claim 1, wherein the embryonic stem cells are cultured in a medium in the presence of at least one agent or cytokine in order to differentiate into specific cells or tissues.

3. The method of claim 2, wherein the agent or cytokine is selected from the group consisting of IL-1, TNF-a, IL-6, PTH, PDGF, PGE 2 cAMP, estrogens, anti-estrogens, progestins, anti-progestins, cortisol, GH, androgens, I 3 /T 3 VGEF and cyclosporin.

4. The method of claim 2, wherein the agent or cytokine is present in the culture medium at a concentration of about 1.0 pg/ml to about 10.0 ng/ml.

5. The method of claim 2, wherein the specific cells are selected from the group consisting of nerve cells, bone cells, immune cells, and pancreatic beta cells.

6. The method of claim 2, wherein the embryonic stem cell differentiation is identified by at least one marker substance that accumulates in culture medium.

7. The method of claim 6, wherein the marker substance is selected from the group consisting of an iron sequestering substance, insulin, dopamine, myeloid fibers, and hemoglobin. 8

8. The method of claim 1, wherein the embryonic stem cells are cultured in a medium in the presence or absence of varying concentrations of calcium.

9. A method of propagating a line of pluripotent embryonic stem cells in vivo that originates from either morulae or blastocyst comprising transplanting teacher cells into an area sufficiently close to the embryonic stem cells to allow for at least one agent or cytokine made by the teacher cells to contact the embryonic cells. Dated this 30th day of October 2002 15 MEDICAL COLLEGE OF HAMPTON ROADS S: By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\MaraR\Kee\SpeCi\P391 7 6.doc 30/10/02