Patent Application: US-15956705-A

Abstract:
a method to induce es cells to the cardiac phenotype is disclosed whereby avian precardiac endoderm used as feeder / inducer cells induce high percentage conversion of mouse embryonic stem cells into cardiac myocytes . upon induction , the majority of co - cultured es cell - derived embryoid bodies become enriched in cardiac myocytes and exhibit rhythmic contractions . when precardiac mesoderm is included with the precardiac endoderm , ˜ 100 % of ebs become rhythmically contractile . the inductive effect of the precardiac endoderm / mesoderm is mimicked by medium conditioned by these cells . within each eb induced by medium conditioned by precardiac endoderm / mesoderm , over 80 % of the cells become cardiac myocytes . the inductive efficacy of medium conditioned by avian precardiac endoderm / mesoderm provides a platform to biochemically define factors that induce cardiac myocyte differentiation in es cells , and provides a platform for developing other methods for directing the development of particular cells from stem cells .

Description:
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 .” fig1 shows a general embodiment of a method according to the invention . this figure depicts the three phases of the method : ( 1 ) expansion phase , ( 2 ) suspension phase , and ( 3 ) induction phase . during the expansion and suspension phases , spontaneous differentiation of the stem cells is strategically prevented . induction culminates in terminal cardiac differentiation . 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 fig4 . moreover , cell - free medium conditioned by pree + m ( pree + m - cm ) retains the cardiogenic potency of the explants . the latter findings demonstrate that contact between inducing and responding cells is not required for cardiac differentiation , and that all cardiac myocytes are derived from es cells since there are no embryonic explants in the cultures containing conditioned medium . 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 fig2 ). 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 , fig6 a ), or , cultured in medium previously conditioned by explanted precardiac endoderm + mesoderm cells ( pree + prem - cm ; see tabulated results , fig6 b ). the selection of pre - ebs from the suspension phase for this purpose is restricted , such that the pre - ebs are approximately 200 μm (± 30 μm ) in diameter , thereby containing approximately 1 , 000 cells . adherence to this criterion results in optimal cardiac morphogenesis approximately seven days after the onset of induction , as defined by the development of coherent embryoid bodies that contain multiple foci of rhythmically contractile cardiac myocytes . 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 ( fig6 ). however the chronology of differentiation induced by pree + m - cm is delayed when compared with induction by whole cells ; for example , biochemical differentiation and onset of beating in pre - ebs are respectively observed , on average , six and nine days after induction with pree + m - cm , in comparison with four and seven days after induction with explants . finally , although the increased potency of pree + m , in comparison with pree alone , to induce cardiogenesis in mes cells is unexplained , this result is consistent with findings indicating that stem cell differentiation toward specific cell - type endpoints is influenced by local environments . 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 fig6 ) ebs induced by pree + m or by its conditioned medium ( pree + m - cm ) is unprecedented . using an approach based on induction by an endoderm cell line ( end - 2 cells ) that did not include cells of mesodermal origin or bona fide embryo cells , it has been reported that beating myocytes appeared in only in 35 % of co - cultures , and , that each beating locus contained only 10 - 200 cardiac myocytes . and , although an enrichment of cardiac myocytes from es cells of ˜ 70 % has been obtained , this was achieved by physical selection rather than by embryonic ( direct ) induction . ( xu c . et al . : “ characterization and enrichment of cardiomyocytes derived from human embryonic stem cells .” circ res ., vol . 91 , 2002 , pp 501 - 508 .) hence , the present method uniquely exploits constitutive signals of embryology to achieve a high degree of cardiac myocyte enrichment ( over 80 %) in es cells , as obtained by inducing cardiac myocytes with the same embryonic cells that induce the heart in the embryo . 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 ; 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