Patent Application
20050281791
The present invention relates to protocols to induce stem cells. Specifically, the invention relates to a method for induction of cardiac myocytes in embryonic stem (ES) cells, utilizing mechanistic cues from the embryonic processes that normally regulate the development of the heart in the embryo.
The potential of adult and embryonic stem (ES) cells to regenerate adult tissue has caused extraordinary interest in their therapeutic application. Adult stem cells, residing within niches of mature tissues and in bone marrow are, respectively, considered unipotent and multipotent with regard to their ability to form various cell types. Many instances of adult stem cells' ability to differentiate and become incorporated into adult tissues have been previously disclosed. By contrast ES cells, which are derived from the inner cell mass of blastocyst-stage embryos, are pluripotent, due to their potential to differentiate into all of the more that 200 cell types present in adults.
The therapeutic application of ES cells to regenerate damaged or diseased adult tissues is of high interest. However, such therapeutic utilization requires the resolution of formidable issues including the prevention of immune rejection, assurance that ES cells do not contain infectious agents, and the ability of ES cells to become highly differentiated into the cell type one wishes to repair. Hence the ability to differentiate ES cells to desired cell types is paramount.
The ability to regenerate damaged myocardium with new cardiac myocytes is a major objective of cardiac care, not only to minimize damage following acute pathological cardiac events but also to treat chronic cardiac insufficiency. Hence, generating cardiac myocytes from ES cells has therapeutic appeal. Normally, human and mouse ES cells spontaneously differentiate into cardiac myocytes; unfortunately however, the percentage of spontaneously differentiated cardiac myocytes in populations of ES cells is unacceptably low (˜10%), not only due to their presence in insufficient numbers to effect repair but also due to the high incidence (˜90%) of non-cardiac cells, some of which may form tumors. Therapeutic utilization of ES cells demands that they must be differentiated into cells of the desired target tissue at levels approaching 100%, in order to maximize regenerative efficacy while minimizing potential for tumor formation. Despite the high level of scientific activity in this area of endeavor, to date, no one has achieved direct induction of cardiac myocytes from ES cells via factors that induce heart development in the embryo.
U.S. Pat. No. 6,818,210 describes methods for cellular grafting in myocardial tissue of an animal, comprising forming a stable graft of embryonic cardiomyocyte cells. It also describes a method to manufacture essentially pure populations of cardiac myocytes from ES cells that may be used for such grafts. This method utilizes a positive selection in which ES cells are engineered such that upon their spontaneous differentiation into cardiac myocytes, they manufacture a neomycin-resistance protein that confers resistance to an antibiotic which kills non-myocyte cells in the same culture.
U.S. patent application No. 20050037489 describes a method of generating cells displaying at least one characteristic associated with a cardiac phenotype. The method involves (a) partially dispersing a confluent cultured population of human stem cells, thereby generating a cell population including cell aggregates; (b) subjecting the cell aggregates to culturing conditions suitable for generating embryoid bodies; (c) subjecting the embryoid bodies to culturing conditions suitable for inducing cardiac lineage differentiation in at least a portion of the cells of said embryoid bodies, said culturing conditions suitable for inducing cardiac lineage differentiation including adherence of said embryoid bodies to a surface, and culture, medium supplemented with serum, thereby generating cells predominantly displaying at least one characteristic associated with a cardiac phenotype. A drawback of the described method is that it relies on direct selection of spontaneously-appearing ES-derived cardiac myocytes, does not provide a high level of homogeneity of the cardiac myocyte population, and defines cardiac myocytes as cells exhibiting “at least one characteristic associated with a cardiac phenotype.”
U.S. patent application No. 20040106095 discloses cultured human embryonic stem cells that form embryoid bodies, some of which contain spontaneously-appearing cardiac myocytes that, based on electrical characteristics, represent the major types of cardiac myocytes (ventricular, atrial, nodal) present in the heart. However, methods of inducing homogeneous populations of highly differentiated cardiac myocytes are not addressed. The application describes phenomena occurring only in those myocytes which spontaneously appear under standard culture conditions in some embryoid bodies.
U.S. patent application No. 20040033214 describes methods of purifying pluripotent embryonic-like stem cells and compositions, cultures and clones thereof. The publication also describes a method of transplanting the pluripotent stem cells into a mammalian host, such as human, by introducing the stem cells into the host. The publication also describes to methods of producing mesodermal, endodermal or ectodermal lineage-committed cells by culturing or transplantation of the pluripotent embryonic-like stem cells. Application No. 20040033214 addresses “embryonic-like” stem cells, which are actually adult stem cells. Although the issue of whether such “embryonic-like” adult stem cells may be differentiated into cardiac myocytes is peripherally addressed, issues concerning these cells' homogeneity, and the efficiency with which they become fully differentiated into beating myocytes, are not addressed. Moreover, application No. 20040033214 does not utilize cues based on embryological factors for inducing cellular differentiation.
A recent report by Xu et al. describes use of a density gradient to isolate spontaneously-appearing cardiac myocytes from embryoid bodies cultivated under standard conditions. By this means of direct selection, a 70%-enriched population of ES-derived cardiac myocytes was achieved. The major drawback to this approach is that, in addition to the laborious task of manipulating cells on a density gradient, a large number of embryoid bodies is required due to the small percentage of spontaneously-arising myocytes within each embryoid body.
The invention provides a method to improve the incidence of cardiac myocyte differentiation in a fashion that overcomes drawbacks of existing methods.
A drawback of U.S. Pat. No. 6,818,210 is the requirement for positive selection for pre-engineered ES cells. Advantageously, the present invention provides a method to directly induce a highly purified population of ES-derived cardiac myocytes which does not involve positive selection or the use of antibiotics. Rather, the present invention provides a method by which ES-derived cardiac myocytes are obtained via direct induction with normally occurring factors secreted by the same embryonic tissues that induce heart development in the embryo.
Alternative to using physical separation methods to isolate spontaneously differentiating myocytes, the invention utilizes direct induction to obtain a homogeneous population of ES-derived cardiac myocytes. Benefits of direct induction used according to the method of the invention include acquiring significantly larger numbers of myocytes than can be obtained by direct selection, using a method that is significantly less laborious. The technique of direct induction disclosed herein is based on the mechanism of cardiac myocyte induction which normally occurs during development of the heart within the embryo. The inventors have demonstrated that gastrulation-stage anterior lateral endoderm from chick embryos, hereafter termed ‘precardiac endoderm’ (preE), potently induces cardiac myocyte differentiation in embryonic precardiac mesoderm; subsequently the extraordinary cardiogenic potency of preE was shown by its ability to induce differentiation of cardiac myocytes within non-precardiac mesoderm. The examples herein demonstrate that, when used as a feeder layer, chick preE and/or its associated tissues induce cardiac myocyte differentiation in co-cultured mouse embryonic stem (mES) cells. Specifically, when co-cultured with preE, approximately 65% of embryoid bodies exhibit expression of cardiac markers followed by rhythmic contractility. Most remarkably, it is furthermore disclosed that co-culture of ES cells with preE plus precardiac mesoderm (preM), or replacement of this combination of explants with cell-free medium conditioned by it, respectively induces rhythmic contractility in 100% or 92% of EBs; importantly, the latter are highly enriched (˜86%) in cardiac myocytes.
The invention provides a method to utilize cues, as informed from mechanisms of development which normally occur in the embryo, to direct induction. Toward this end, cues from mechanisms that regulate the normal embryonic development of tissues that would be desirably regenerated in the adult should assist in the design of approaches to induce ES cells into specific cell types.
Another advantage of the present invention is that the resulting cardiac myocytes express a cardiac myocyte marker (i.e. cardiac myosin heavy chain) and also demonstrate rhythmic, synchronous contractility, such physiologic activity indicating the expression and subsequent organization of tens to hundreds of proteins which must cooperate to establish a functional cardiac myocyte.
The present invention also uses normally occurring inductive factors to achieve unanimously contractile embryoid bodies containing a highly purified (>80%) population of cardiac myocytes.
The invention provides a method that can be used with efficacy both in vitro and in vivo.
The invention provides a method to demonstrate that cell-cell contact is not necessary for the cardiogenic effect induced by secretions of precardiac endoderm+mesoderm.
Furthermore, the disclosed methods provide a framework or platform for extending this approach to include the induction of ES cells into the cellular phenotypes of organs other than the heart, using rationales based on the cues provided by the respective embryonic mechanisms which regulate the development of said cell types in the embryo.
The invention provides a method for the induction of cardiac myocytes from embryonic stem cells comprising the following steps: (i) expanding cultures of ES cells to approximately 60% confluency in a defined medium; (ii) dispersing said stem cells in suspension culture conditions to provide suspended cell aggregates; (iii) growing cell aggregates to form pre-embryoid spheres (preEBs); (iv) selecting said preEBs of a size within an optimal size range of about 170-230 μm; and (v) subjecting said selected preEBs to culturing conditions suitable for induction of cardiac myocytes.
It is described here for the first time that embryonic stem (ES) cells can be induced to form cardiac myocytes with high levels of enrichment for rhythmically contractile myocytes and that the method of the invention that induces such cardiac myocyte differentiation also inhibits spontaneous differentiation of ES to other cell types. We call this invention “Cardiomyogenesis on Cue.”
It is known that cardiac myocytes can be found in embryoid bodies formed from ES cells, having arisen by spontaneous differentiation,. In such embryoid bodies, stem cell differentiation into a variety of tissue types typically occurs. In the absence of molecular guidance provided by using cues akin to those that regulate natural development during embryology, the spontaneous incidence of cardiac myocytes occurs at a therapeutically disappointing low number (i.e. ˜10% of total cells in the embryoid body).
By using the same cells that induce the heart in an embryo, the present method achieves a very high degree of cardiac myocyte enrichment in ES cells. There are a number of known techniques that give rise to the formation of embryoid bodies, and, the methods used for forming the EBs result in a greater or lesser percentage of EBs containing some cardiac myocytes. According to the invention, exploitation of normal developmental cues vastly improves the incidence of cardiac myocytes, in a predictable manner of time and yield. Because this technique indicates an approach to obtain homogeneous populations of cardiac myocytes from ES cells, clinical application of the latter may be facilitated.
The potency of embryonic precardiac endoderm (preE), but not posterior endoderm (postE), to induce terminal cardiac myocyte differentiation in co-migrating precardiac mesoderm cells in the embryo is known. According to the invention, the inclusion of precardiac mesoderm with endoderm (preE+M) strongly increases the incidence of cardiogenesis in ES cells, causing substantially all EBs to become rhythmically contractile. The effect of co-culturing pre-EBs with precardiac endoderm (preE), which specifies and induces differentiation of precardiac mesoderm in the embryo, is shown in
An embodiment of the method comprises three steps—expansion, suspension, and induction—the latter culminating in terminal cardiac myocyte differentiation. During expansion and suspension phases, the method prevents spontaneous differentiation of mES cells, so that cardiac differentiation can be induced on cue during the induction phase.
To prevent spontaneous differentiation during the expansion phase, ES cells are expanded on a layer of MEF cells in MEF-conditioned medium which contains no LIF (leukemic inhibitory factor) but which does contain FGF-2 (fibroblast growth factor-2). Importantly, LIF, which is conventionally used to prevent spontaneous differentiation of ES cells, is withheld because this factor inhibits cardiac myocyte differentiation. And, FGF-2 was included because this factor was deemed to (i) inhibit differentiation of pluripotent cells and (ii) promote cardiac myocyte differentiation once this process is induced by other unknown factors. According to the invention ES cells are grown to no greater than 60% confluence during the expansion phase, since increasing confluency (i.e. the extent of cellular density in the culture) promotes spontaneous differentiation.
To initiate the suspension phase, the same growth conditions are used except that the MEF feeder layer and the fibronectin coating on the culture dish surface are removed. As a result, ES cells continue to grow during their literal suspension in the growth medium. Because the ES cells cannot adhere to the culture dish surface, they adhere to each other, resulting in the formation of multicellular spheres termed “pre-embryoid bodies”. To achieve optimal cardiac myocyte differentiation during the next (induction) phase, the suspension phase must last for only a maximum of 2-3 days (in contradistinction to 7+days as employed in standard protocols), and, that pre-EBs selected for the induction phase must not exceed about 200 μm (±15%) diameter (see
To initiate the induction phase during which cardiac myocyte differentiation is induced, suspended pre-EBs are individually implanted adjacent to, or directly upon, explanted avian precardiac endoderm (preE) and/or mesoderm (preM) cells (see tabulated results,
Cell to cell contact is not required to induce cardiac myocyte differentiation, as demonstrated by the ability of cell-free medium conditioned by explanted endoderm and mesoderm to mimic the inductive effect of explanted cells (
In addition to its ability to induce heart development in the embryo, precardiac endoderm (preE) can induce cardiogenesis at ectopic sites in the embryo, indicating that preE has a role in the ‘specification’ of cells to the cardiac lineage, which in the embryo occurs during early gastrulation. It is believed that at the onset of gastrulation, as ingressing cells from the epiblast become diverged into endodermal and mesodermal germ layers, the endoderm specifies precardiac mesoderm within a subset of the mesoderm germ layer. It is further believed that these embryonic processes are mimicked during the conversion of pre-EBs to beating EBs when, under the influence of signals from preE+M that are sufficiently potent to over-ride competing signals that may be present to induce the differentiation of other cell types, cardiac myocytes are induced in highly enriched fashion.
The observation of rhythmic contractility in virtually all (100%; see
The present method to induce mouse ES cells to a cardiac phenotype utilizes culture conditions that can be seamlessly applied to human ES cells. Most significantly, LIF (leukemic inhibitory factor) is omitted from, and FGF (fibroblast growth factor) is added to, mouse embryonic fibroblast (MEF)-conditioned medium during the expansion and suspension phases to prevent spontaneous ES cell differentiation.
It is known that treatment of human ES cells with combinations of purified growth factors induces phenotypes having characteristics of specific embryonic germ layers; however, EBs enriched for single cell types are not generated. On the other hand, treatment of less primitive embryonic cells, from gastrulation and post-gastrulation stage embryos, with specific growth factor combinations is known to induce specific cell types including pituitary, neural crest, and cardiac myocytes (the latter having been discovered by the inventors). Rationally defined cocktails of purified growth factors, based on composition of the environment in which embryonic cells differentiate, may induce enriched cohorts of specific cell types from ES cells. The cardiogenic potency of preE+M, combined with the demonstration that preE+M can be replaced by its conditioned medium, provides a starting point for the biochemical identification of these cells' secretory products, with the goal of defining a cocktail of purified growth factors that can induce cardiogenesis in ES cells on cue.
This Application claims priority from U.S. provisional patent application Ser. No. 60/581,946 filed Jun. 22, 2004.
This invention was made with United States government support from the National Institutes of Health (NIH) grant numbers NIH HL39829-16A1S1 (JL) and NIH T 32 HL07792 (DRR). The United States government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60581946 | Jun 2004 | US |
