EP1265987A2 - Populations de cellules pluripotentes et de cardiomyocytes, moyens d'obtention et utilisations - Google Patents

Populations de cellules pluripotentes et de cardiomyocytes, moyens d'obtention et utilisations

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Publication number
EP1265987A2
EP1265987A2 EP01916536A EP01916536A EP1265987A2 EP 1265987 A2 EP1265987 A2 EP 1265987A2 EP 01916536 A EP01916536 A EP 01916536A EP 01916536 A EP01916536 A EP 01916536A EP 1265987 A2 EP1265987 A2 EP 1265987A2
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Prior art keywords
cardiomyocytes
conduction
promoter
cells
cell
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EP01916536A
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German (de)
English (en)
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Loren J. Field
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Indiana University Research and Technology Corp
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Indiana University Research and Technology Corp
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Publication of EP1265987A2 publication Critical patent/EP1265987A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates generally to the field of cardiology, and in particular to cardiomyocyte cell populations, and methods for obtaining and using the same.
  • the conduction system of the heart determines heart rate in response to signals from the nervous system as well as chemical signals delivered from other organs.
  • the conduction system provides impulses to the myocardium in a coordinated fashion, which impulses facilitate the coordinative action of various sections of the heart to pump blood.
  • This conduction system is supported by the regular generation of a depolarization wave which causes contraction of the myocardium.
  • the coordination of the location and frequency of the depolarization wave is critical in rendering the heart responsive to its own needs as well as other needs of the body.
  • pacing the heart has been developed, in which a stimulus is supplied to the heart to generate a depolarization wave and attendant contraction of the heart.
  • an electromechanical pacemaker to apply regular stimuli to the heart to facilitate a regular heartbeat in patients lacking sufficient natural heart pacing function.
  • an electrode of the pacemaker is implanted in myocardial tissue at the apex of the right ventricle, and the remaining pacemaker components are implanted under the patient's skin, typically near the chest .
  • U.S. Patent No. 5,103,821 describes an attempt to formulate a protocol for a biological pacemaker.
  • the '821 patent proposes to harvest sino-atrial node cells from an area of the heart in the right atrium where the cells are located. Removal of these cells is accomplished by catheters which include small biopsy devices which remove a small quantity of cells but do not penetrate the right atrium. The next step in the '821 process is to culture the harvested sino-atrial node cells to generate a larger quantity of cells.
  • the cultured cells are then introduced into the right ventricle of the heart using an implant catheter, in particular at the apex of the right ventricle, a location which experience with electromechanical pacemakers has demonstrated is advantageous for initiating the polarization waves.
  • the '821 patent proposes that this process will provide the patient with a biological pacemaker which functions almost identically to the natural pacing of the heart.
  • a difficulty with the '821 proposal is that experience has shown that the culture of adult cardiomyocytes is not a straightforward endeavor, and in fact to date the applicant knows of no suitable methods for culturing adult cardiomyocytes as would enable the conduct of a procedure as proposed in the '821 Patent.
  • the conduction system of the heart is known to involve at least three cell types, including sino-atrial node cells, atrial-ventricular node cells, and Purkinje cells.
  • the sino-atrial node is the primary heart pacemaker and is located at the junction of the superior caval vein and the right atrium. Immunohistochemical studies demonstrate that sino-atrial node cells can clearly be differentiated from atrial myocytes, each with its own characteristic phenotype. P.W. Oosthoek et al . , Circulation Research, Vol. 73, No. 3, pp. 473-481 (1993).
  • the atrial-ventricular node is composed of small myocytes embedded in connective tissue.
  • the atrial-ventricular node terminates in fingerlike myocytes that make contact with myocytes of the atrial-ventricular bundle, and Purkinje cells form a network of Purkinje fibers in the myocardium which deliver excitation current to the working myocardium.
  • P.W. Oosthoek et al. Circulation Research, Vol. 73, No. 3, pp. 482-491 (1993).
  • research into the development of the heart including its conduction system has involved in vi tro differentiation of embryonic stem cells. For example, it has been observed that embryonic stem cells differentiate into cells with properties of the sinus node, atrium, and ventricle.
  • V.A. Maltsev et al . Circulation Research, Vol. 75, No. 2, pp 233-244 (March 1994); A.M. Wobus et al . , Annals New York Academy of Sciences 752, pp 460-469 (March 1995) .
  • Another area of cardiologic research concerns the diagnosis and treatment of damaged myocardial tissue to restore contractile function to the heart.
  • Several conditions injure tissue composed of working atrial or ventricular cardiomyocytes.
  • Such conditions include, for instance, tissue necrosis stemming from ischemic conditions such as coronary blockage myocardial infarction or the like.
  • ischemic conditions such as coronary blockage myocardial infarction or the like.
  • efforts have been made to develop satisfactory therapies to re-establish contractile activity lost due to such injuries, they have been hindered by the generally non-regenerative character of the patient's existing cardiomyocytes .
  • the invention provides a cellular population comprising cardiomyocytes and which is enriched in conduction cardiomyocytes relative to working cardiomyocytes.
  • cardiomyocytes Preferably, of the cardiomyocytes in the population, at least 50% are conduction cells, e.g. sino-atrial and/or atrial- ventricular node cells.
  • Such a population may be obtained by a method of the invention, which comprises (i) providing a multipotent cell such as an embryonic stem cell which differentiates to cardiomyocytes, the multipotent cell including a conduction cardiomyocyte selection marker which enables selection of conduction cardiomyocytes from other cardiomyocytes; (ii) causing the multipotent cell to differentiate; and, (iii) selecting the conduction cardiomyocytes based on the selection marker.
  • the selection marker can enable a positive or a negative selection protocol, with illustrative selection markers including antibiotic resistance genes and the HSV t ymidine kinase gene.
  • a single-step selection protocol in which the desired conduction cardiomyocyte population is obtained in a single selection
  • a multi-step selection protocol e.g. involving a stem cell with introduced DNA enabling an initial selection of a general cardiomyocyte population including conduction cardiomyocytes and working cardiomyocytes, and also enabling a subsequent selection of a conduction cardiomyocyte-enriched population from the general cardiomyocyte population.
  • the present invention also provides a transgenic multipotent (also known as pluripotent) cell such as an embryonic stem cell, which includes DNA having a selection gene fused to a promoter, wherein the DNA enables selection of a cell population enriched in conduction cardiomyocytes relative to working cardiomyocytes.
  • a transgenic multipotent cell such as an embryonic stem cell
  • Such stem cells may be constructed by transfecting an embryonic stem cell with a vector including the DNA.
  • Such methods and vectors also constitute embodiments of the present invention.
  • the invention also concerns a method for pacing a heart in a mammal, which includes establishing in the heart a stable cellular graft of fetal cardiomyocytes including node cells, wherein the node cells pace the heart.
  • the present invention provides an animal model useful for cardiologic study.
  • the invention provides a non-human mammal having a heart and a stable graft of fetal cardiomyocytes in the heart, wherein the cardiomyocytes are enriched in node cells relative to other cardiomyocytes .
  • Another aspect of the invention concerns a method for restoring function to an injured regional segment of the myocardium of a mammal .
  • the method includes implanting viable cardiomyocytes in the regional segment .
  • the invention also concerns a device which includes a substrate adapted for in vivo implantation into myocardial tissue, and viable cardiomyocytes coated on the substrate.
  • a preferred device is a conductive lead, for example a pacemaker lead.
  • the device may also be an elongate filament, such as a suture.
  • the substrate may be coated directly with the cardiomyocytes, or the device may include a carrier coated on the substrate, for instance a gel, carrying the viable cardiomyocytes .
  • the present invention provides cardiomyocyte and multipotent cell populations, and methods for obtaining and using the same.
  • the invention also provides related vectors, animal models and engraftment procedures for heart pacing and restoration of contractile activity, as well as improved articles for heart implantation coated with viable cardiomyocytes.
  • the invention thereby provides improvements both in the implementation of conventional electromechanical devices, and in avoiding or supplementing the function of such electromechanical devices .
  • the present invention features enriched populations of fetal conduction cardiomyocytes and methods and materials for obtaining and using the same.
  • the fetal conduction cardiomyocytes utilized in the present invention will have the electrophysiologic properties consistent with pacemaker cells which act to establish depolarization waves in the heart.
  • the conduction cardiomyocytes will generally demonstrate automaticity (i.e. spontaneous contractile activity) such as that displayed by sino-atrial (SA) node cells and/or atrial-ventricular (AV) node cells.
  • SA sino-atrial
  • AV atrial-ventricular
  • Substantially enriched populations of conduction cardiomyocytes can be obtained for use in the invention by the differentiation of a genetically engineered multipotent cells, illustratively embryonic stem (ES) cells, somatic stem cells, or other multipotent cells, having means for selecting conduction cardiomyocytes from other cardiomyocytes to which the stem cells differentiate, such as working atrial and ventricular cardiomyocytes .
  • ES embryonic stem
  • Preferred embryonic stem or other multipotent cells of the invention will thus include introduced DNA encoding a selection marker fused to a promoter, which provides for (positive or negative) selection of conduction cardiomyocytes from other cardiomyocytes.
  • a selection marker conferring increased survivability to a selection pressure is fused to a promoter which causes expression of the selection marker in the cell type to be selected, but not in cell types to be eliminated.
  • a positive selection gene such as a gene conferring resistance to an antibiotic or other lethal agent, is fused to a promoter which causes expression of the gene in conduction cardiomyocytes but not in other cardiomyocyte types. Subjecting a differentiated population containing the conduction and other cardiomyocytes to the corresponding selection pressure, e.g. the antibiotic, will then provide a selected population which is enriched in viable conduction cardiomyocytes relative to the initial population.
  • a selection gene which . decreases survivability of the cell in the presence of a selection pressure.
  • This selection gene is fused to a promoter causes its expression in cell types other than those to be selected, but not in the cells to be selected.
  • the mixed population is then subjected to the selection pressure to provide a cell population which is enriched in the selected cell types relative to the initial population.
  • HSV-TK HSV thymidine kinase
  • an embryonic stem cell or other multipotent cell can be provided carrying a negative selection gene such as the HSV thymidine kinase gene fused to a promoter which is inactive in conduction cardiomyocytes but active in other cardiomyocytes, for example the connexin43 promoter (Oosthoek et al . , Circulation Research, Vol. 73, No. 3, pp. 473-481 (1993); Oosthoek et al . , Circulation Research, Vol. 73, No. 3, pp. 482-491 (1993)).
  • a negative selection gene such as the HSV thymidine kinase gene fused to a promoter which is inactive in conduction cardiomyocytes but active in other cardiomyocytes, for example the connexin43 promoter (Oosthoek et al . , Circulation Research, Vol. 73, No. 3, pp. 473-481 (1993); Oostho
  • the negative selection gene e.g. the HSV thymidine kinase gene
  • the negative selection gene will not be expressed in the conduction cardiomyocytes, which will thus not be susceptible to the negative selection agent, e.g. gancyclovir.
  • the negative selection gene will be expressed in other cardiomyocytes (e.g. working cardiomyocytes) , which will thus be susceptible to the negative selection agent, e.g. gancyclovir.
  • Incubation of the mixed cardiomyocyte population containing the conduction and other cardiomyocytes in the presence of the negative selection agent will kill the other cardiomyocytes, but not the conduction cardiomyocytes. A conduction cardiomyocyte-enriched population is thereby obtained.
  • Selection methods of the invention may include one selection step or multiple selection steps, so long as at least one selection step enriches the population in conduction cardiomyocytes relative to other cardiomyocytes .
  • An illustrative one-step selection may be provided with an embryonic stem cell containing a positive selection gene (e.g. an antibiotic resistance gene) fused to a promoter which is specific to conduction cardiomyocytes in the context of the selection protocol (i.e. the promoter is active essentially only in conduction cardiomyocytes and not any other cell type differentiated from the embryonic stem cell at the time of selection) .
  • This stem cell is differentiated to provide a population including the conduction cardiomyocytes and other cells, which population is subjected to the positive selection pressure (e.g.
  • Illustrative positive selection genes which may be used for these purposes include the neomycin and hygromycin resistance genes.
  • Illustrative promoter candidates for these purposes include promoters specific to or enhanced in conduction cardiomyocytes including, for instance the minK promoter.
  • an embryonic stem or other multipotent cell may be provided having means for selecting a first population differentiated from the stem cell, and for selecting a second population from the first population.
  • the embryonic stem or other multipotent cell may, for instance, carry means for selection of a general cardiomyocyte population including conduction and other cardiomyocyte types, and then for selecting a conduction cardiomyocyte-enriched population from the general cardiomyocyte population.
  • the multipotent cell may carry a positive selection gene such as an antibiotic resistance gene fused to a promoter active in cardiomyocytes but not other cell types.
  • Suitable such promoters include for instance the beta-MHC promoter, and can be used to prepare highly- purified fetal cardiomyocyte populations as reported in WO 95/14079 (26 May 1995) .
  • the multipotent cell can also- be engineered to contain a negative selection gene fused to a promoter which is inactive in conduction cardiomyocytes but active in other cardiomyocytes.
  • Suitable negative selection genes include for instance the HSV-TK gene.
  • Suitable candidate promoters for these purposes include for example the connexin43 promoter.
  • Such a stem cell can then be differentiated to provide an initial cell population containing cardiomyocytes and other cell types, and the positive selection pressure applied to select a general cardiomyocyte population. Thereafter, the negative selection pressure can be applied to the general cardiomyocyte population to select the conduction cardiomyocyte-enriched population .
  • the minK promoter may be used to facilitate obtaining node cell-enriched populations. It has been reported that minK promoter is restricted to or at least enhanced in the conduction system of the heart. Kupershmit et al . , Circulation Research, Vol. 84, No. 2, pp. 146-152 (February 1999) . This promoter is fused to a selection gene such as the amino glycoside phosphotransferase fusion gene to permit G418 selection of differentiated cultures of embryonic stem or other multipotent cells as generally described above. After establishing a clonal undifferentiated embryonic stem or other multipotent cell line, the cells are differentiated in vi tro . G418 selection is imposed once beating cells (i.e. cardiomyocytes) are observed.
  • beating cells i.e. cardiomyocytes
  • conduction cardiomyocytes are selected by this in vi tro differentiation process and enables a determination of the extent of enrichment in conduction cardiomyocytes. For example, conduction cardiomyocytes and working ventricular and atrial cardiomyocytes can be discerned from one another by differing action potential profiles and/or patterns of connexin expression.
  • a multipotent cell such as an embryonic stem cell including a alpha-MHC promoter fused to a neomycin resistance gene, and also including a fusion gene comprised of the connexin43 promoter fused to the (HSV-TK) gene.
  • cardiomyocytes in general are selected by incubation with G418. Afterward, a second round of selection with gangcyclovir is initiated. Cells expressing the HSV-TK gene will incorporate the gangcyclovir and die, whereas cells not expressing the HSV- TK gene will survive.
  • connexin43 promoter is active in the ventricular and atrial working cardiomyocytes, these cells will die. In contrast, the conduction cardiomyocytes do not express connexin43, will not express the HSV-TK gene and thus will survive gangcyclovir selection. Again, the extent of conduction cardiomyocyte enrichment in the selected cultures can be ascertained using molecular and electrophysiologic analyses .
  • Multipotent cell-derived conduction cardiomyocyte- enriched populations obtained as described above or fetal cardiomyocytes can be used for example to generate biologic pacemakers in living mammals.
  • this involves the implantation of the multipotent cell-derived or fetal conduction cell-containing population into the heart tissue of a mammal (including human patients) in need of heart pacing, wherein the implanted population paces the heart .
  • a mass of SA node cells located on the right atrium is responsible for pacing the heart in a normally-functioning heart. This mass is responsible for generating an initial depolarization wave which ultimately results in the contraction of the entire heart muscle and in particular the heart muscle forming the wall of the right ventricle and the wall of the left ventricle.
  • the SA node cells initiate such a depolarization wave which spreads through the muscle tissue of the right atrium until the wave front arrives at another small knot of cells in the right atrium identified as the atrial ventricular (AV) node.
  • the AV node is located low in the rear wall of the right atrium and passes downwardly within a wall which separates the right ventricle from the left ventricle.
  • Heart disease can result in impaired function of the SA node or can damage or interrupt the atrium surrounding the AV node, so that depolarization does not reach the AV node. This interrupts the depolarization wave to the left and right ventricles. In other impairments, fractures may occur in the Bundle of His or branches therefrom with the result that the depolarization wave generated by the SA node does not reach the right and left ventricles in a uniform and efficient fashion. In still other cases, complete heart blockage of the depolarization wave can occur.
  • the ES-derived or fetal conduction cells may be implanted in the heart in order to restore or improve the function of the heart in terms of pacing.
  • the conduction cells may be implanted within the native SA node or AV node structures, or elsewhere in the conduction system, to supplement the functions of these structures.
  • a pacing graft of the conduction cells may be located in a separate region of the heart.
  • the pacing graft may be located in regions found to be effective for implantation of the lead of a conventional electromechanical pacemaker.
  • this may include a location in the right ventricle of the heart, especially at or near the extreme apex of the right ventricle.
  • Such location has been found with electromechanical pacemakers to provide an effective site for stimulation to achieve efficient contractile activity of the heart for pumping blood.
  • This or other locations of the right ventricle are selected so as to generate a depolarization wave from the fetal or ES-cell- derived conduction cell graft which radiates in a uniform and orderly progression along the walls of the right and left ventricles .
  • the engraftment procedure may involve catheterization of the heart so as to implant the cells at or near the internal surfaces of the heart. Alternatively, the cells may be engrafted on the exterior surface of the heart in an open or potentially laproscopic procedure.
  • a number of injections or implantations of cells may be necessary to provide pacemaker activity of the graft in larger mammals such as humans.
  • Anticipated total graft sizes in humans are from about 10 4 to about 10 7 cells or more.
  • These established, viable grafted cells can be provided by one or multiple cellular implantations, e.g. by implanting up to about 10 8 or more cells at a time.
  • the graft is established by multiple implantations (e.g. injections) of up to about 250,000 cells at a time. If necessary after a preliminary operation, one or more additional operations can be conducted to add to the size of the graft to optimize its biological pacing or other activity.
  • neo-innervation in a pacemaker graft can be achieved by targeting the expression of growth factors which promote neo-innervation, for example nerve growth factor (NGF) (see, e.g., U.S. Patent Nos . 5,082,774; 5,169,762; 5,272,063; and 5,288,622).
  • NGF nerve growth factor
  • an NGF expression cassette is introduced into skeletal myoblasts (C2C12 cells) and/or fibroblasts, stably transformed cell lines are established, and the capacity to produce biologically active NGF is ascertained in an in vi tro neurite outgrowth assay.
  • C2C12 cells C2C12 cells
  • fibroblasts stably transformed cell lines are established, and the capacity to produce biologically active NGF is ascertained in an in vi tro neurite outgrowth assay.
  • the genetically-modified cells are engrafted into an adult syngeneic host.
  • the animals are sacrificed, and harvested and processed for immunohistologic analyses.
  • NGF fusion gene expression is monitored by in si tu and anti-NGF immunohistologic analyses.
  • conduction cardiomyocyte-enriched populations can themselves deliver proteins such as nerve growth factor, or can be co-engrafted with other cells delivering such proteins, to optimize the establishment and performance of the biological pacemaker graft.
  • neo-innervation may be monitored by non-invasive techniques, for instance by PET scanning utilizing a suitable imaging agent such as 11- C-hydroxy epheddrine.
  • Negative controls in such experimentation include grafts lacking the engrafted cell type (e.g. myoblasts or fibroblasts) , as well as grafts with the engrafted cell type but lacking the NGF transgene .
  • retrograde labeling techniques can be used to determine the coupling of the pacemaker graft to the cardiac ganglia. Subsequent physiological experiments can be used to confirm that the pacemaker graft is subject to autonomic regulation in instances where AV node activity is blocked pharmacologically and/or physically.
  • the present invention also concerns cellular engraftment procedures which are used to restore function to regions of the myocardium which have suffered injury resultant, for example, of regional tissue injury stemming from ischemia, apoxia, hypoxia or the like.
  • tissue injury stemming from ischemia, apoxia, hypoxia or the like.
  • the blockage or restriction of a coronary artery such as by a blood clot, can lead to injury of a particular area of the heart served by the artery due to inadequate blood supply.
  • the area of injury may involve impairment or death of the myocardial tissue in the region.
  • Such conditions cause a decrease in the contractile function of the heart and in the efficiency with which it pumps blood.
  • an engraftment procedure using cardiomyocytes or other suitable cells may be conducted to restore function to the region of injured myocardium.
  • Preferred restorative grafts of the invention will be located at or adjacent to the region of injured myocardium, and will desirably be introduced as soon as possible after the infarct or other injury, during active granulation tissue formation but prior to scarring and myocardial thinning.
  • Preferred grafts will include cardiomyocyte cells which are physically and electronically coupled to the viable native myocardium adjacent to the injured myocardium. Such coupling can be observed, for example, by the organization of the engrafted cells and the formation of nascent junctional complexes both between engrafted cells themselves and between engrafted cells and the native cardiomyocytes. Several implantations of cardiomyocytes may be necessary to provide the restorative properties to the graft.
  • the graft is established by multiple implantations (e.g. injections) of up to about 250,000 cells at a time to achieve a graft size of about 10 4 to 10 7 cells or more.
  • at least one implantation of cells will be made such that the engrafted cells contact viable myocardial tissue for instance on the perimeter of the injured region of myocardium.
  • multiple injections or implantations of cells may be made around the periphery of the injured region so as to substantially surround the injured region with engrafted, viable cells.
  • cells from a prior engraftment may be used as the newly established periphery, and grafts may be constructed so as to provide viable, coupled cardiomyocytes substantially into or substantially throughout the injured myocardial region, so as to provide the improved functionality to the region.
  • the patient can be monitored for improvement in contractile function of the heart and the loss or decrease in functional artifacts caused by the injured region of myocardium.
  • Such monitoring can be conducted by signal processing methodologies which are known and used in the detection and localization of ischemic or other injured myocardium.
  • the invention provides means to generate a cellular interface between a conductive lead and the host myocardium, e.g. for an electromechanical pacemaker, defibrillation device, or the like.
  • a conductive lead e.g. for an electromechanical pacemaker, defibrillation device, or the like.
  • traditional pacemakers activate ventricular depolarization through a bipolar lead which field stimulates a region of the endocardial surface in the apex of the ventricular. Because the electrical signal is dissipated through 360°C, and because there is frequently fibrosis between the lead and the excitable myocardium, a significant amount of energy is needed to trigger cardiac depolarization.
  • a cellular interface is constructed between the conductive lead and the excitable myocardium.
  • the invention provides a conductive lead which includes a carrier impregnated with viable cardiomyocytes.
  • the carrier can for example be a gel such as a collagen gel.
  • the lead can by any lead known for heart implantation, including for instance straight, helicle, barbed and other lead types.
  • Figure 1 shows one such lead.
  • device 11 adapted for fixed implantation into the heart tissue.
  • Device 11 includes conductive lead 12 coated with a carrier layer 13.
  • Carrier layer 13 can be formed of any suitable carrier material which maintains the viability of the cardiomyocytes.
  • Preferred carriers will be gels, for example collagen gels. Contained in carrier layer are viable cardiomyocytes 14.
  • the conductive lead is electrically connected to an electromechanical device 15, e.g. a pulse generator of a pacemaker, via wire 16.
  • the tip of the lead 12 including the carrier 13 and cardiomyocytes is inserted into the myocardium, facilitating coupling between the viable cardiomyocytes 14 and the excitable myocardium, and the electromechanical device 15 is conventionally implanted in the patient.
  • the pacemaker discharge from the pulse generator 15 does not have to traverse a region of fibrosis (or has to traverse only a smaller region of fibrosis) , and because the discharge is not dissipated through a 360°C area, stimulation of the interface cardiomyocytes 14 can be accomplished with lower energy utilization.
  • Excitation of the host myocardium is actuated through nascent junctional complexes formed between the host and interface cardiomyocytes (preferably fetal cardiomyocytes) .
  • the use of such cellular interfaces between pacemaker leads can for example reduce the energy needed to stimulate ventricular depolarization as compared to the methods currently used for field stimulation. This in turn can extend the useful battery life in current systems.
  • FIG. 2 shown is another article of the invention 20 which includes an elongate filament 21, for example a suture, having viable cardiomyocytes 22 coated thereon.
  • Article 20 optionally may also include a carrier layer 23 (shown in phantom) to facilitate adherence of the cardiomyocytes 22 to the filament 21; however, it has been found that cardiomyocytes have a natural tendency to adhere directly to surfaces, even without the carrier.
  • Article 20 is useful, e.g., in the in vi tro study of the electrophysiologic properties of cardiomyocytes 22, including their ability to transmit a signal along the length of the filament 21.
  • Article 21 may also be used in vivo by implanting article 21 into myocardial tissue to similarly study signal transmission and/or to modify the existing pattern of initiation and/or propagation of depolarization waves in the heart.
  • article 20 may be implanted into the myocardium so as to span between two regions, and thus provide a filamentous cellular graft therebetween.
  • Such graft may then serve to modify signal transmission between the two regions, participating for instance in the enhancement of the propagation of a depolarization wave between the two regions to improve the efficiency of heart pumping.
  • the cell-populated filament may be implanted to span from the SA nodal region to the left ventricular wall, e.g. in the treatment of dilated cardiomyopathy.
  • This Example details selection protocols for obtaining cell populations enriched in cardiomyocyte sub-lineages, including node cells. It has been shown that a cardiac lineage-specific promoter (i.e. the alpha-cardiac myosin heavy chain promoter) fused to a drug resistance gene (i.e. a cDNA encoding aminoglycoside phosphotransferase) can be used to select essentially pure cultures of cardiac myocytes in differentiating cultures of ES cells (see U.S. Patent No. 5,602,301 and WO 95/14079, each hereby incorporated by reference) .
  • a cardiac lineage-specific promoter i.e. the alpha-cardiac myosin heavy chain promoter
  • a drug resistance gene i.e. a cDNA encoding aminoglycoside phosphotransferase
  • a fusion gene comprising the MLC2v promoter driving expression of a cDNA encoding aminoglycoside phosphotransferase is constructed.
  • the fusion gene also contains a phosphoglycerate kinase (pGK) -hygromycin cassette.
  • Undifferentiated ES cells are transfected with the MLC2v-neo/pGK-hygro fusion gene. Transfected cells are initially selected and cloned by virtue of their resistance to hygromycin. The resultant clonal lines are subsequently differentiated in vi tro, and subjected to G418 selection when cardiomyocyte differentiation is apparent (i.e. once beating cells are detected) .
  • the resulting population of selected cells is examined for cardiac- and ventricular- specific markers to ascertain he degree of lineage- specificity in the selected cell population. Markers examined include ANF, MLC2a, MLC2v, a-MHC, ⁇ -MHC and cTnT. Identification of cells with attributes consistent with ventricular cardiomyocytes, but not atrial and conduction cardiomyocytes, or relatively fewer atrial and conduction cardiomyocytes establishes that a cardiomyocyte cell population enriched in ventricular cardiomyocytes can be selected by this in vi tro differentiation process.
  • Transgenic mice are generated with this construct using conventional methodology to ascertain the cell-type specificity of cardiac minK promoter.
  • Mice resulting from embryos injected with the minK- ⁇ GAL construct are screened to identify transgenic founders. These animals are bred, and the pattern of ⁇ GAL activity is monitored in fetal, neonatal and adult transgenic offspring. These experiments establish the temporal cardiac-specific pattern of expression.
  • this promoter is used too for selection in ES-derived cardiomyocytes.
  • the promoter is incorporated into an aminoglycoside phosphotransferase fusion gene to permit G418 selection of differentiating cultures of ES cells.
  • Molecular and electrophysiological criteria are used to establish the degree of conduction system selection.
  • a transgene comprised of an ⁇ -MHC promoter fused to a neomycin resistance gene is used to select a mixed population of cardiomyocytes of atrial, ventricular and conduction system origin.
  • This system is expanded to a double selection scheme to eliminate the non- conduction system cells.
  • the connexin43 promoter is used, which is active in working ventricular and atrial cardiomyocytes, but not in node cells.
  • a fusion gene comprised of the connexin43 promoter and the Herpes Simplex Virus thymidine kinase gene (HSV-TK) is introduced into ES cells carrying the MCH-neo r construct.
  • HSV-TK Herpes Simplex Virus thymidine kinase gene
  • cardiomyocytes are selected by incubation with G418. A second round of selection with gangcyclovir is then initiated.
  • Cells expressing the HSV- TK gene incorporate the gangcyclovir and die, whereas cells not expressing the HSV-TK gene survive.
  • the connexin43 promoter is active in the ventricular and atrial working cardiomyocytes, these cells will die.
  • the node cells do not express connexin43, will not express the HSV-TK gene and thus survive gangcyclovir selection.
  • Molecular and electrophysiologic analyses are performed to ascertain the extent of conduction cell enrichment in these selected cell cultures.
  • connexin 40 is expressed in the Purkinje cells but not in working cardiomyocytes (Oosthoek, P.W., S. Viragh, W.H. Lamers, and A.F. Moorman. Immunohistochemical delineation of the conduction system. II: The atrioventricular node and Purkinje fibers. Circulation Research . 73: 482-491, 1993; Oosthoek, P.W., S. Viragh, A.E. Mayen, M.J. van Kempen, W.H. Lamers, and A.F. Moorman. Immunohistochemical delineation of the conduction system. I: The sinoatrial node. Circulation Research .
  • Transgenes are generated which utilize the connexin 40 promoter (Seul, K.H. , P.N. Tadros , and E.C. Beyer. Mouse connexin40: gene structure and promoter analysis. Genomics 1997. Nov. 15. 46: 120- 126, 1998) to drive expression of a neomycin resistance expression cassette.
  • the construct is built into a pGH- hygro r vector backbone.
  • ES cells are transfected with the Connexin 40-neo r /pGK-hygromycin transgene. 24 hrs later, the cells are subjected to hygromycin selection.
  • the cultures are induced to differentiate. Once beating cells are detected (8 days post-induction) , the cultures are subjected to G418 selection. Once selected cultures are in hand, the degree of cardiomyocyte enrichment is ascertained. In preliminary experiments, G418-selected cultures are digested with trypsin and replated onto collagen coated chamber slides at a density suitable for immune cytologic analysis of individual cells. 24 hours post plating, the cells are screened for sarcomeric myosin cardiomyocyte enrichment when using atrial-restricted promoters.
  • a series of cardiac engraftment experiments demonstrate the usefulness of cellular grafts as a biological pacemaker.
  • Cell populations enriched in node cells are selected as described in Example IB, IC or ID and directly engrafted into the left ventricle of a syngeneic adult mouse. Cardiac automaticity is monitored throughout the experiment in vivo using surface electrocardiographic recordings.
  • Three weeks post-engraftmen the heart is harvested and placed on a Langendorf/working heart apparatus. Functional hearts can be maintained in vi tro for three to four hours on this apparatus .
  • the anatomic origin of pacemaker activity is monitored by ECG leads placed on the epicardial surface.
  • Heart block is induced pharmacologically (via perfusion) and/or surgically (via cautery) of the AV node.
  • the capacity of the engrafted cells to drive ventricular depolarization is monitored through the epicardial ECG leads .
  • Variables including optimal cell number to provide pacemaker activity (as determined by a resident ⁇ GAL reporter in the conduction cells) and anatomic position of the graft are ascertained in this system.
  • the genetically modified C2C12 cells are engrafted into an adult syngeneic host.
  • the animals are sacrificed and the hearts harvested and processed for immunohistologic analyses.
  • NGF fusion gene expression is monitored by in si tu and anti-NGF immunohistologic analyses.
  • the extent of neo-innervation is assessed using immunologic markers for both sympathetic and parasympathetic neurons, as well as by histologic examination and measurements of cardiac catecholamine content.
  • Retrograde neuron labeling experiments are initiated with working heart preparations in vi tro to track the anatomic origins of the nascent neurons within the heart. In all instances, myoblast grafts lacking the NGF transgene are employed as negative controls.
  • NGF-expressing cells are co-engrafted with selected pacemaker cells. These experiments determine the level at which NGF expression facilitates innervation of the pacemaker cells within the ectopic graft site.
  • immunohistologic analyses determines the extent of neo- innervation. Negative controls include grafts lacking myoblasts, as well as grafts with myoblasts lacking the NGF transgene.
  • a cellular interface is constructed between the pacemaker lead wire and the excitable myocardium. This would consist of an insulated wire which terminates with a collagen gel plug impregnated with cardiomyocytes, for example fetal cardiomyocytes obtained from differentiating ES cells as described herein and/or in WO 95/14079.
  • the tip of this lead is inserted into the myocardium, facilitating coupling between the cardiomyocytes and the excitable myocardium. Because the pacemaker discharge does have to traverse a region of fibrosis (or has to traverse only a smaller region) , and because the discharge will not be dissipated throughout a 360° area, stimulation of the interface ES- derived cardiomyocytes is accomplished with much lower energy utilization. Excitation of the host myocardium is actuated through nascent junctional complexes formed between the host and interface cardiomyocytes.
  • segment length shortening was normalized to pre-dobutamine value for each sonomicrometer pair according to the following formula:
  • ⁇ SS [(post dobutamine SS - pre-dobutamine SS)/pre- dobutamine SS] x 100.
  • ES- or fetal-derived Purkinje cells are in hand, we will generate an "artificial Purkinje fiber".
  • a series of experiments will be performed to optimize the seeding procedure. This will include a number of straight forward experiments which will test the timing of seeding post differentiation, the effects of varying the concentration of cell, optimization of the suture material (i.e. collagen coated, fibronectin coated, etc.), and optimization of culture agitation (rotating vs. rocking, rate, etc.).
  • the experimental endpoints consist of measuring the percentage of the suture which is successfully seeded, as well as determine the cell thickness on the suture.
  • the capacity of the suture to propagate action potentials is also tested. In first pass screens this is easily accomplished by simply monitoring contraction of the cells along the suture. In addition, quantitative monitoring of the action potential propagation is performed using voltage sensitive dyes. Microelectrode records are also performed. If necessary, cells are placed at the edge of the structure with an extracellular electrode and monitor action potential propagation.
  • seeded sutures are generated which are able to propagate action potentials, they are used in experiments designed to synchronize contraction between primary cardiomyocyte cultures in adjacent chambers on a multichamber slide. Towards that end, the seeded suture are draped across the dam dividing the two chambers, and primary cardiomyocytes are seeded in each chamber . The dam is modified (lowered) so as to allow the suture to be completely immersed in culture media. After the cells are attached and exhibit automaticity, cells in one chamber are paced (using extracellular electrodes) at a rate in excess of the spontaneous rate of the cultures. Extracellular electrode records are obtained simultaneously in cells in the non-paced chamber.
  • seeded suture If the seeded suture is able to propagate the action potential, cells in the non-paced chamber should contract at a rate identical to those in the paced chamber.
  • the establishment of successful generation of seeded sutures suitable to drive pacing in vitro will lead to experiments to determine if cells present on the seeded suture are viable in vivo.
  • a short segment of seeded suture is affixed to the surface of a syngeneic adult mouse heart. The ends of the suture are inserted into the epicardial surface (by "burying" the end into a small hole produced with a 30 gauge syringe needle) .
  • the central region of the suture is allowed to lie freely on the epicardial surface of the heart.
  • the heart is removed and the viability of the cells on the suture is monitored by histologic analyses. Any inflammatory responses to the presence of the sutures is monitored by standard histochemistry (H and E, Masson's trichrome) and immune histology (anti-macrophage anti- leukocyte, etc.).

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Abstract

L'invention concerne des populations cellulaires enrichies en cardiomyocytes de conduction, ainsi que des procédés et des matériaux nécessaires à leur obtention. Ces populations peuvent être utilisées pour faire prendre une greffe de tissu myocardique mammalien, par exemple dans la mise en place de stimulateur cardiaque biologique. L'invention concerne aussi des greffes myocardiques cellulaires restauratrices destinées à améliorer la fonction contractile de segments du myocarde endommagés, ainsi que des articles conçus pour être implantés dans le coeur (par exemple des électrodes de stimulateur cardiaque), recouverts de cardiomyocytes viables et comprenant, éventuellement, un support pour les cardiomyocytes.
EP01916536A 2000-03-10 2001-03-12 Populations de cellules pluripotentes et de cardiomyocytes, moyens d'obtention et utilisations Withdrawn EP1265987A2 (fr)

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US7732199B2 (en) 2001-07-12 2010-06-08 Geron Corporation Process for making transplantable cardiomyocytes from human embryonic stem cells
CN1543500B (zh) 2001-07-12 2014-04-09 杰龙公司 从人多能干细胞产生心肌细胞系细胞
EP1453382A4 (fr) 2001-11-08 2005-05-18 Univ California Methodes et compositions de correction des troubles de la conduction cardiaque
US7638330B2 (en) 2003-03-21 2009-12-29 Gabriella Minchiotti Method of promoting the differentiation of staminal cells
US7452718B2 (en) 2004-03-26 2008-11-18 Geron Corporation Direct differentiation method for making cardiomyocytes from human embryonic stem cells
EP1745144B1 (fr) 2004-05-11 2010-12-01 Axiogenesis Ag Dosage pour la decouverte de medicament reposant sur des cellules differenciees in vitro
WO2006021459A1 (fr) * 2004-08-27 2006-03-02 Cell Center Cologne Gmbh Compositions et methodes destinees a la modulation de la differenciation cellulaire
KR101529317B1 (ko) 2005-06-22 2015-06-16 아스테리아스 바이오세라퓨틱스, 인크. 영장류 다능성 줄기 세포의 심근세포 계통 세포로의 분화
CN100422739C (zh) * 2006-04-13 2008-10-01 东南大学 便携式缺氧操作箱
WO2008126083A2 (fr) * 2007-04-11 2008-10-23 Technion Research & Development Foundation Ltd. Procédés d'identification et de sélection de cellules dérivées de cellules souches embryonnaires humaines
SG188098A1 (en) 2008-01-30 2013-03-28 Geron Corp Synthetic surfaces for culturing stem cell derived cardiomyocytes
WO2012098260A1 (fr) 2011-01-21 2012-07-26 Axiogenesis Ag Système non viral pour générer des cellules souches pluripotentes induites (ips)
US9592398B2 (en) 2011-05-12 2017-03-14 Medtronic, Inc. Leadless implantable medical device with osmotic pump
WO2017192602A1 (fr) 2016-05-02 2017-11-09 Emory University Utilisations d'inhibiteurs de la transition épithélio-mésenchymateuse dans la génération de cellules pacemaker

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US5103821A (en) * 1989-03-06 1992-04-14 Angeion Corporation Method of providing a biological pacemaker
US5602301A (en) * 1993-11-16 1997-02-11 Indiana University Foundation Non-human mammal having a graft and methods of delivering protein to myocardial tissue
US6127176A (en) * 1998-06-10 2000-10-03 Cleveland Clinic Foundation Mutant cell lines unresponsive to interleukin 1

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WO2001068814A2 (fr) 2001-09-20
CA2402245A1 (fr) 2001-09-20

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