Patent Application
20100278787
The present invention relates to novel cardiomyocyte-like cell clusters (CMLC) derived from hBS cells and to the potential use of such cardiomyocyte-like cell clusters in e.g., pharmaceutical drug discovery and development, toxicity testing, cell therapy and medical treatment. The clusters contain cells expressing endodermal, mesodermal as well as cardiac markers. The invention also relates to a method for preparing the cluster, to composition comprising one or more CMLC for use in therapy and toxicity testing. The compositions are stable after storage of the composition at a temperature of at least −80° C. for at least 2 years, i.e. the characteristics and viability of the clusters are not substantially changed during this storage period.
Mature cardiomyocytes are considered terminally differentiated cells and as such they have no, or very low, proliferative capacity. Human cardiomyocytes can be isolated from heart biopsies but the procedure is complicated and it is difficult to obtain viable cell preparations. In addition, the access to human heart tissue is very limited and it is thus not possible to isolate large number of cells. An easy access to cells with a phenotype of human cardiomyocytes is critical for the development of cell therapy interventions for cardiac disease. In addition, due to the lack of donor material as well as the somewhat problematic procedure of cell isolation, human primary cardiomyocytes are not currently available for in vitro testing during pre-clinical drug discovery.
Populations of pluripotent human stem cells can be isolated from the inner cell mass of blastocysts and these cells have the capacity for indefinite, undifferentiated proliferation in vitro (Thomson et al., 1998; Reubinoff et al., 2000; Heins et al., 2004). Differentiation of human blastocysts derived stem (hBS) cells may occur spontaneously in vitro, especially during, for undifferentiated cells, sub-optimal culture conditions (Thomson et al., 1998; Reubinoff et al., 2000). In addition, hBS cells can be coaxed to differentiate in a directed fashion along specific pathways forming a variety of specialized cell types including cardiomyocytes, endothelial cells, neuronal cells, insulin producing β-cells, and hematopoietic cells (reviewed in: Keller, Genes Dev 2005). However, relatively little is currently known about how to control and manipulate hBS cell differentiation to produce exclusive populations of specific cell types.
For the derivation of cardiomyocytes from hBS cells, in principle two different procedures have been reported. The first is through differentiation of hBS cells initiated when the cells are cultured in suspension and form embryoid bodies (EBs) (Itskovitz-Eldor et at 2000, Kehat et al 2001). Within these mixed population of cells contracting areas with functional properties of cardiomyocytes can be observed. The second procedure is based on co-culture of hBS cells with END-2 cells (a visceral endoderm-like cell line) which results in the formation of beating clusters of cells that also display characteristics of cardiomyocytes (Mummery et al 2003, Passier et al 2005). Other variants of the described protocols included forced aggregation (Burridge et al 2007) and the hanging drop procedure (Yoon et al 2006). Based on the variations of the protocols used and the efficiency at which hBS cells differentiate to cardiomyocytes, it appears that hBS cell lines behave quite differently, implicating that the actual hBS cell line of use might affect the final result. Thus, each cell line may require a specific differentiation protocol for induction of cardiogenesis.
During recent years, a number of papers have described, in various ways, the basic characteristics of hBS cell-derived cardiomyocytes. In these reports, cell analysis has been based on the expression of molecular markers for cardiomyocytes, structural architecture, and functionality (reviewed in: Ameen et al 2007 Crit Rev Oncol Hematol). The morphology and ultrastructure of hBS cell-derived cardiomyocytes share similarities with adult cardiomyocytes although the myofibrillar and sarcomeric organization indicate an immature phenotype in the stem cell derived population (Kehat et al 2001, Snir et al 2003, Norstrom et al 2006, Yoon et al 2006). On a molecular level, several markers expressed by cardiomyocytes are also expressed by hBS cell-derived cardiomyocytes, including transcription factors, structural proteins, hormones, ion-channels, and tight junction proteins (Kehat et al 2001, Xu et al 2002, Mummery et al 2003, He et al 2003, Passier et al 2005, Norstrbm et al 2006, Yoon et al 2006).
Taken together, the molecular and structural properties of the hBS cell derived cardiomyocytes suggest that these cells share similarities with their adult counterparts. More importantly, however, are the functional characteristics of the cells, and different pharmacological and electrophysiological approaches have been used to examine these properties. One major advantage of cardiomyocytes derived from hBS cells is that they can be maintained in culture for extended time periods without loosing their spontaneous contractile capacity. This allows for repeated non-invasive examination of the same cell preparations. Several studies have demonstrated that hBS cell-derived cardiomyocytes respond to α/β-adrenergic- and muscarinic stimuli suggesting that the cells express specific surface membrane receptors coupled to a signalling pathway that activate ion channels, membrane transporters and myofilament proteins (Kehat et al 2001, Xu et al 2002, Xue et al 2005, Norstrom et at 2006). In addition, action potentials indicative of nodal-, atrial- and ventricular-like origin have been identified in hBS cell-derived cardiomyocytes using intracellular electrophysiological measurements (Xu et al 2002, Mummery et at 2003, He et al 2003). Taken together, these results indicate that in vitro developed stem cell derived cardiomyocytes have a basal functionality which makes them attractive for further evaluation in terms of applicability in drug discovery.
Being a tissue like structure, cardiomyocyte like cell clusters from hBS cells specifically, has a great potential for several of these in vitro applications were they make up a platform for measurements of functional attributes such as action potentials and conduction in cardiomyocytes. In addition, the developing clusters may serve as in vitro models to study early events during human cardiogenesis. Furthermore, based on the tissue similarities the clusters may be optimal for use in vivo for restoration of cardiac function after injury or disease. The present invention presents unique cardiomyocyte-like cell clusters derived from hBS cells for use according to the above.
The present invention relates to novel cardiomyocyte-like cell clusters (CMLC) and the methods for their preparation from hBS cells. The CMLC of the present invention are especially well suited for use in drug discovery and toxicity testing.
A cluster according to the invention comprises cardiomyocyte-like cells, wherein the cluster has
i) contracting cells,
ii) cells that are electrically connected,
and expresses
iii) cardiac markers including Nkx.2.5, troponin and myosin,
iv) markers for functional adrenergic receptors,
v) markers for functional muscarinic receptors,
vi) markers for functional ion-channels including hERG, Na+, Ca2+ and K+ channels,
vii) one or more endodermal markers selected from the group consisting of AFP, TF, APOA2, AHSG, SERPINA1, APOA1, APOC3, TTR, APOB, and RBP4
The electrical connection between cells mentioned above may be by gap junctions.
In a specific embodiment, said cluster does not express one or more of the following markers for undifferentiated cells: OCT-3/4, SSEA-4, TRA-1-60 as described in example 13.
It is important that the cluster contains cells that respond to pharmacological stimuli such as beta-adrenergic stimulation (isoproterenol, adrenalin), alpha-adrenergic stimulation (phenylephrine), muscarinic stimulation (carbachol, acetylcholine, atropine), blockage of calcium channels (verapamil), blockage of hERG channels (E4031) and inhibition of funny channels.
A cluster according to the present invention comprises cardiomyocyte-like cells and the cluster comprising genes that are up-regulated and have,
i) expression values of 500 or more,
ii) a fold change in gene expression between cardiomyocyte-like cell clusters and undifferentiated hBS cells (FCCMLC) of 10 or more.
In the examples herein are demonstrated suitable methods for the determination of the above-mentioned values.
The present inventors have found that cardiomyocyte-like cells suitable for use in e.g. medical applications, drug screenings and cardiotoxicity studies can be isolated in the form of clusters that are relatively easy to prepare in a reproducible manner and without any need for further differentiation into cardiomyocyte cells. Besides cardiac markers, the clusters also contains markers e.g. for endodermal lineage, express one or more ion channels commonly used for cardiotoxicity testing, and express transcription factors.
Accordingly, the present invention provides a relative simple means of obtaining a suitable cardiomyocyte-like cell product for medical use as well as for drug screening and testing without the need of purification and complete differentiation to mature cardiomyocytes.
Moreover, the present invention provides a composition that presents the CMLC in a form that is easy to use for the end-user. The composition is prepared by suspending the CMLC in an aqueous medium notably containing agents that ensure the stability of the CMLC during freezing/vitrification, storage, thawing and use of the cluster. Accordingly, the properties of such an aqueous medium must be chosen in consideration of all these four different processes in order to obtain a sole medium that is suitable for the handling of the clusters from the clusters have been prepared and until use of the end-user. The product is presented in fresh or frozen form.
As used herein, the term “blastocyst-derived stem cell” is denoted BS cell, and the human form is termed “hBS cells”. In literature the cells are often referred to as embryonic stem cells, and more specifically human embryonic stem cells. The pluripotent stem cells used in the present invention can thus be embryonic stem cells prepared from blastocysts, as described in e.g. WO 03/055992 and WO 2007/042225, or be commercially available hBS cells or cell lines. However, it is further envisaged that any human pluripotent stem cell can be used in the present invention, including differentiated adult cells which are reprogrammed to pluripotent cells by e.g. the treating adult cells with certain transcription factors, such as OCT4, SOX2, NANOG, and LIN28 as disclosed in Junying Yu, et al., 2007.
As used herein the term “DMSO” is intended to mean dimethylsulfoxide.
As used herein the term “cardiomyocyte-like cells” is intended to mean cells sharing features with mature cardiomyocytes. Cardiomyocyte-like cells are further defined by morphological characteristics as well as by specific marker characteristics.
As used herein the terms “cardiomyocyte-like cells clusters” (CMLC) is intended to mean a group comprising at least two cardiomyocyte-like cells attached to each other, and which also comprise other cell types. A cluster contains beating cells.
As used herein the term “nodal-like cells” is intended to mean cells with the following action potential characteristics:
Membrane resting potential (MRP): −40 to −60 mV,
Duration: 80-130 ms, and
A pronounced phase 4 depolarization.
As used herein the term “atrial-like cells” is intended to mean cells with the following action potential characteristics:
Membrane resting potential (MRP): −50 to −80 mV, and
Duration: <150 ms.
As used herein the term “ventricle-like cells” is intended to mean cells with the following action potential characteristics:
Membrane resting potential (MRP): −50 to −80 mV,
Duration: >150 ms, and
A pronounced plateau phase.
In the present context, the term “expression” is used for gene and/or protein expression, whatever must be relevant in the context.
In the present application, the term “cryopreservation” denotes the preservation of biological material at an extremely low temperature.
In the present context, the term “mixed differentiated hBS cells” is used for a population of spontaneously differentiated hBS cells were cells from all three germ layers mesoderm, endoderm and ectoderm are present. This sample is used as control material to distinguish genes up regulated in many types of differentiated cells from genes specifically up regulated in cardiomyocyte-like cell clusters. In the examples herein is given instructions how to obtain mixed differentiated hBS cells.
In the present context, the term “forced aggregation” is used for 3D aggregation of cells by an external force, such as centrifugation with a centrifugal force or sedimentation by gravity.
Feeder Cells
As used herein feeder cells are intended to mean supporting cell types used alone or in combination. The cell type may further be of human or other species origin. The tissue from which the feeder cells may be derived include embryonic, fetal, neonatal, juvenile or adult tissue, and it further includes tissue derived from skin, including foreskin, umbilical chord, muscle, lung, epithelium, placenta, fallopian tube, glandula, stroma or breast. The feeder cells may be derived from cell types pertaining to the group consisting of human fibroblasts, fibrocytes, myocytes, keratinocytes, endothelial cells and epithelial cells. Examples of specific cell types that may be used for deriving feeder cells include embryonic fibroblasts, extraembryonic endoderm cells, extraembryonic mesoderm cells, fetal fibroblasts and/or fibrocytes, fetal muscle cells, fetal skin cells, fetal lung cells, fetal endothelial cells, fetal epithelial cells, umbilical chord mesenchymal cells, placental fibroblasts and/or fibrocytes, placental endothelial cells,
As used herein, the term “MEF cells” is intended to mean mouse embryonic fibroblasts.
As used herein, the term “TGF-β” means transforming growth factor beta, preferably of human and/or recombinant origin. TGF-β is a protein that comes in three isoforms called TGF-β1, TGF-β2 and TGF-β3. The TGF-β family is part of a superfamily of proteins known as the transforming growth factor beta superfamily, which includes inhibins, activin, anti-müllerian hormone, bone morphogenetic protein, decapentaplegic and Vg-1.
As used herein, the term “FGF” means fibroblast growth factor, preferably of human and/or recombinant origin, and subtypes belonging thereto are e.g. bFGF (sometimes also referred to as FGF2) and FGF4.
The term “BMP2” is intended to mean bone-morphogenic protein-2 which is one member of the BMP family of growth factors.
As used herein the term “xeno-free” is intended to mean complete circumvention of direct or in-direct exposure to non-human animal components.
Cardiac Stem Cell Marker:
c-kit and cardiac progenitor markers: Islet-1, Nkx2.5, GATA-4. Here regarded as early cardiac markers.
Markers for cardiomyocytes: cTnl, cTnT (cardiac troponin I and T), α-MHC (alpha-myosin-heavy-chain), connexin-43.
Other markers appear from the examples herein.
CMLC
A cluster according to the invention comprises cardiomyocyte-like cells, wherein the cluster has
i) contracting cells,
ii) cells that are electrically connected,
and expresses
iii) cardiac markers including Nkx.2.5, troponin and myosin,
iv) markers for functional adrenergic receptors,
v) markers for functional muscarinic receptors,
vi) markers for functional ion-channels including hERG, Na+, Ca2+ and K+ channels,
vii) one or more endodermal markers selected from the group consisting of AFP, TF, APOA2, AHSG, SERPINA1, APOA1, APOC3, TTR, APOB, and RBP4
The electrical connection between cells mentioned above may be by gap junctions.
A cluster of the invention preferably does not express one or more of the following markers for undifferentiated cells: OCT-3/4, SSEA-4, TRA-1-60. In a specific embodiment the cluster does not express any markers for undifferentiated cells.
In an aspect the present invention provides a cluster comprising cardiomyocyte-like cells, the cluster comprising genes that are up-regulated and have,
i) expression values of 500 or more,
ii) a fold change in gene expression between cardiomyocyte-like cell clusters and undifferentiated hBS cells (FCCMLC) of 10 or more.
The above mentioned values can be measured as described in Example 2 herein. The present inventors have applied two specific ways of obtaining the fold change values:
The results from the two methods differ slightly and it is contemplated that the use of method ii), i.e. where a further selection is performed, may lead to a cluster having properties more suitable for use in specific applications.
Accordingly, in a specific embodiment, the present invention provides a cluster comprising cardiomyocyte-like cells, the cluster comprising genes that are up-regulated and have,
i) expression values of 500 or more,
ii) a fold change in gene expression between cardiomyocyte-like cell clusters and undifferentiated hBS cells (FCCMLC) of 10 or more, and
iii) a ratio between FCCMLC and FCMC of 100 or more.
FMC denoted the fold change for a mixed differentiated hBS cell population. In the present context, the term “mixed differentiated hBS cells” is used for a population of spontaneously differentiated hBS cells where cells from all three germ layers mesoderm, endoderm and ectoderm are present. This sample is used as control material to distinguish genes up-regulated in all types of differentiated cells from genes specifically up-regulated in cardiomyocyte-like cell clusters.
Furthermore, the expression values are obtained by measurement with a specific chip (GeneChip® Human Genome 0133 Plus 2.0 (Affymetrix Inc., Santa Clara, Calif., USA) as detailed described in Example 2 herein. Use of other chips may give different values and therefore any comparison with the values herein should be made using the same kind of chip or a chip giving similar results.
In a more specific embodiment of the invention, a cluster is thus provided wherein the cluster comprising genes which are up-regulated and have,
i) expression values of 2000 or more,
ii) a FCCMLC value of 100 or more, and
iii) a ratio between FCCMLC and FCMC of 100 or more.
In the examples herein details are given with respect to how a person skilled in the art can carry out experiments in order to determine the above-mentioned values.
The clusters are derived from BS cells, notably hBS cells. Accordingly, the starting material may be commercially available BS cells or cell lines or, alternatively, the starting material may be prepared as described in WO 03/055992 and WO 2007/042225. Such material can be obtained from Cellartis AB (www.cellartis.com) and is also available through the NIH stem cell registry (http://stemcells.nih.gov/research/registry) as well as from the UK Stem Cell Bank (http://www.ukstemcellbank.org.uk).
As mentioned before, not every individual cell contained in the cluster may fulfill the above-mentioned requirements. The requirements are fulfilled for the cluster and not necessarily for any individual cell. Furthermore, the cells contained in the cluster may be of different degree of differentiation; some may be entirely differentiated into cardiomyocyte-like cells, whereas others may possess characteristics like endodermal cells. However, in any event, the cluster must contain contracting cells. As mentioned in the examples herein, the clusters are typically prepared by subjecting BS, notably hBS, cells to spontaneous differentiation via forced aggregation to obtain 3D-structures containing undifferentiated cells. The cardiomyocyte like cell clusters appear after further cultivation of the 3D-structures for up to 22 days.
Furthermore, the different cells and differentiation degree of the cells contained in the cluster is envisaged to be advantageous in the applications of the CMLC. For example, the various cell types may be of importance for increasing adhesion and binding of the CMLC to different surfaces in vitro (e.g. culture dishes or glass surfaces, coated or non-coated). They could also contribute to enhanced cell survival during cryopreservation and improved engraftment of the cells when CMLC are used for in vivo applications (e.g. cell transplantation). Accordingly, an objective of the present invention is to provide a cluster containing different types of cells in their development from hBS cells to cardiomyocytes, i.e. from the very beginning of differentiation via intermediate differentiation to fully differentiated cells into cardiomyocytes. As demonstrated in the examples herein none of the cells in the clusters has been found to be undifferentiated cells. Accordingly, and as demonstrated in the examples and figures, a cluster of the invention has a variety of markers.
Normally, the clusters contain a plurality of cells such as from about 10 to about 5000 cells.
In a specific embodiment, a cluster according to the invention contains cells with up-regulated genes (compared to undifferentiated cells), wherein the expression value is about 750 or more such as, e.g., an expression value of about 1000 or more, about 1500 or more, about 2000 or more, about 2500 or more or about 3000 or more.
Alternatively, or moreover, a cluster according to the invention contains cells with up-regulated genes having a FC(CMLC) value of about 20 or more such as, e.g., about 50 or more, about 100 or more, about 500 or more, about 750 or more, about 1000 or more. Alternatively or additionally (when the method involving mixed differentiated hBS cells) is employed), the ratio of FCCMLC/FCMC is about 15 or more such as, e.g., about 20 or more, about 50 or more, about 100 or more, about 500 or more, about 750 or more, about 1000 or more. A set of criteria can be set in order to select clusters with different content of up-regulated genes.
Some of the genes identified in the clusters are genes that are not normally associated with cardiomyocyte-like cells (see the genes marked as “white” in Table II). These findings seem to be of interest and they may play an important role for the functionality of the CMLC with respect to medical use and testing. Specifically cells and tissue of endodermal origin seem to play an essential role in early cardiac development. The CMLC make up a tissue like structure simulating early developing cardiac tissue.
As shown in the Examples herein, a cluster according to the invention has the following characteristics (Table II and III):
Of particular note is that the majority of the genes in Table II can be classified into two different groups. The first group consists of genes previously associated with cardiac cells and the second of genes previously associated with endodermal cells, such as hepatocytes. Examples of genes belonging to the first group are (marked in light grey in table II): MYH6, MYL7, MYL4, TNNC1, TNNT2, PLN, TTN, MYH7, LDB3, NPPB, GATA6, MYL3, CSRP3, ACTN2, MB, MYOZ2, TBX5, and HSP27. Examples of genes belonging to the second group are (marked in dark grey in table II): AFP, TF, APOA2, AHSG, SERPINA1, APOA1, ALB, APOC3, TTR, APOB, and RBP4.
To get an even stricter selection of genes specifically up-regulated in CMLC the criteria were narrowed to:
The remaining 9 genes are listed in Table III and these represent suitable marker genes for the CMLC.
The selection shows an even distribution between endodermal and mesodermal genes expressed in the clusters. The mesodermal genes and the endodermal are marked in, light grey and dark grey respectively in Table III.
In other specific embodiment, a cluster according to the invention has 2 or more such as, e.g., 4 or more, 6 or more, 8 or more, 10 or more, 12 or more or 16 or more of the up-regulated genes are genes associated with cardiac cells, and/or
2 or more such as, e.g., 4 or more, 6 or more, 8 or more of the up-regulated genes are genes associated with endodermal cells, and/or
2 or more such as, e.g., 4 or more, 6 or more, 8 or more of the up-regulated genes are genes associated with non cardiac or non endodermal cells, described in Table II herein, and/or the up-regulated genes comprise 10 or more such as, e.g., 20 or more, 30 or more, 40 or more, 50 or more, 55 or more or all genes listed in Table II herein.
In a more specific embodiment of the invention a cluster is provided wherein the cluster comprising genes which are up-regulated and have,
In a further embodiment of the present invention a cluster is provided wherein the cluster comprising genes which are up-regulated and have,
The cluster is normally derived from BS cells such as, e.g., hBS cells and the number of cells in the cluster is normally 10 to about 5000 cells or from 10 to about 2000 cells.
Moreover, a cluster according to the invention expresses one or more ion channels. Notably, the ion channels identified are ion channels commonly used in cardiotoxicity testing. This is a further indication of the suitability of the CMLC for cardiotoxicity testing.
The ion-channel is typically a K-, Na, and/or Ca-ion channel such as a K-voltage-gated channel, a K-inwardly-rectifying channel, a Na-voltage-gated channel, a Na-ligand-gated channel, and/or a Ca-voltage-dependent channel. More specific ion channels are listed in
In a very specific embodiment, a cluster according to the invention has all genes listed in any of Table II, Ill and/or listed in
More specific embodiments appear from the appended items and claims.
The clusters of cardiomyocyte-like cells described above may also be characterized by measuring their transmembrane action potential. By using a sharp microelectrode to enter the clusters, as further described in Example 11, the characteristic action potential were recorded and the AP duration at 50%, 70% and 90% of repolarization (dur50 (APD50), dur70 (APD70), dur90 (APD90)), AP amplitude (amp), membrane resting potential (MRP), and maximum rate of rise of the AP upstroke (Vmax) were determined.
The clusters were thus characterized as predominately nodal-like, atrial-like, or ventricle-like based on the results of transmembrane action potential (TAP) recordings. Cluster, or suspension of clusters, were thus found that comprised at least 10%, (such as e.g. at least 13%, at least 15%, or about 17%) of nodal-like cells and/or at least 30% (such as e.g. at least 35%, at least 40%, at least 45% or about 50%) of atrial-like cells and/or at least 20% (such as e.g. at least 23%, at least 26%, at least 30%, or about 33%) ventricle-like cells.
As seen from the Tables above, the clusters contain cells with non-cardiac markers and express a gene number exceeding previously reported studies. Thus, a cluster according to the invention typically comprises cells expressing one or more such as at least 5, at least 10, at least 15, at least 20, at least 25 or all of the following genes:
More specifically, and using the more restricted requirements as mentioned above, a cluster of the invention comprises cells expressing one or more such as at least 2, at least 3, at least 4, at least 5 or all of the following genes:
More details are given in
A suspension or composition of cardiomyocyte-like clusters may contain a mixture of clusters containing nodal-like cells, clusters containing atrial-like cells and clusters containing ventricle-like cells.
One embodiment the invention thus relates to a suspension or composition of cardiomyocyte-like clusters, wherein the ratio between the number of clusters containing nodal-like cells and the number of clusters containing atrial-like cells is in a range of from about 1:100 to about 50:100 such as from about 10:100 to about 40:100, from about 20:100 to about 40:100, from about 30:100 to about 40:100 or about 33:100-34:100.
In another embodiment the invention relates to a suspension or composition of cardiomyocyte-like clusters, wherein the ratio between the number of clusters containing nodal-like cells and the number of clusters containing ventricle-like cells is in a range of from about 1:100 to about 80:100 such as from about 10:100 to about 70:100, from about 30:100 to about 70:100, from about 45:100 to about 55:100 or about 50:100.
In a further embodiment the invention relates to a suspension or composition of cardiomyocyte-like clusters, wherein the ratio between the number of clusters containing ventricle-like cells and the number of clusters containing atrial-like cells is in a range of from about 1:100 to about 90:100 such as from about 40:100 to about 80:100, from about 50:100 to about 75:100 or about 66:100.
In a still further embodiment the invention relates to a suspension or composition of cardiomyocyte-like clusters, wherein the ratio between the clusters containing nodal-like cells, the clusters containing atrial-like cells and the clusters containing ventricle-like cells is 17:50:33.
Compositions Comprising One or More Cluster
In another aspect of the invention relates to a cluster-containing composition, i.e. one or more clusters of cardiomyocyte-like cells as described above may be in a composition comprising a carrier, such as e.g. an aqueous medium. The composition may further contain one or more additives including one or more cryoprotectants, one or more stabilizers and/or one or more viscosity-adjusting agents. The composition may be in liquid or frozen form.
The one or more cryoprotectants may be selected from the group consisting of ethylene glycol, propylene glycol, dimethylsulfoxide, glycerol, propanediol, and methyl pentanediol, and/or mixtures thereof. The additive may also be a sugar or sugar alcohol including sucrose, trehalose, maltose or lactose.
In a special embodiment, the cryoprotectant is trehalose in a concentration from about 0.02 M to about 1 M, such as, e.g., from about 0.05 M to about 0.9 M, from about 0.1 M to about 0.8 M, from about 0.15 M to about 0.7 M, from about 0.2 M to about 0.65 M, from about 0.25 M to about 0.6 M. The concentration of trehalose may preferably be about 0.3 M.
In a another special embodiment the cryoprotectant is sucrose in a concentration from about 0.02 M to about 1 M, such as, e.g., from about 0.05 M to about 0.9 M, from about 0.1 M to about 0.8 M, from about 0.15 M to about 0.7 M, from about 0.2 M to about 0.65 M, from about 0.25 M to about 0.6 M. The concentration of sucrose may preferably be about 0.3 M.
In a further embodiment, the cryoprotectant may be DMSO in a concentration that is at least 2.5% v/v, such as e.g. from about 2.5% to about 40% v/v, from about 5% to about 35% v/v, from about 7% to about 30% v/v, from about 7% to about 25% v/v, from about 7% to about 20% v/v, from about 15% to about 25% v/v, or from about 5% to about 15% v/v.
In a still further embodiment, the cryoprotectant may be ethylene glycol in a concentration that is at least 2.5% v/v, such as e.g. from about 2.5% to about 30% v/v, from about 5% to about 25% v/v, from about 5% to about 20% v/v, from about 10% to about 20% v/v, from about 7% to about 10% v/v, or from about 2.5% to about 5% v/v.
The composition may further comprise a viscosity-adjusting agent selected from the group consisting of Ficoll, Percoll, hyaluronic acid, albumin, polyvinyl pyrrolidone, alginic acid, gelatin and glycerol.
In a special embodiment, the viscosity-adjusting agent may be Ficoll in a concentration at the most about 150 mg/ml, such as, e.g., at the most about 100 mg/ml, at the most about 50 mg/ml, at the most about 25 mg/ml, at the most about 15 mg/ml or at the most about 10 mg/ml.
A further component of the composition may be one or more growth factors, e.g. basic fibroblast growth factor (bFGF) in a concentration of about 2 ng/ml, such as about 3 ng/ml, about 4, ng/ml, about 5 ng/ml, about 6 ng/ml or more.
The composition may also comprise one or more essential and/or non-essential amino acids in appropriate concentrations: Examples of essential amino acids are e.g. L-Alanine, L-Arginine, L-Asparagine, L-Aspartic acid, L-Cysteine, L-Glutamic acid, L-Glutamine, Glycine, L-Histidine, L-Isoleucine, L-Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine in concentrations raging from about 0.01 mM, such about 0.05 mM, about 0.1 mM, about 0.15 mM, about 0.2 mM or more.
The composition may further include one or more inorganic salts such as Calcium chloride (Anhydrous), Cupric sulfate (CuSO4-5H2O), Ferric sulfate (FeSO4-7H2O), Potassium chloride (KCl), Magnesium chloride (Anhydrous), Sodium chloride (NaCl), Sodium bicarbonate (NaHCO3), Sodium phosphate, dibas (Anhydrous), Zinc sulfate (ZnSO4-7H2O), and/or one vitamins, such as Biotin, D-Calcium pantothenate, Choline chloride, Folic acid, i-Inositol, Niacinamide, Pyridoxine hydrochloride, Riboflavin, Thiamine hydrochloride or Vitamin B12.
The above-mentioned substances may also be available in a commercial mixture e.g., DMEM (Dulbecco's Modified Eagle's Medium), MEM media or RPMI media.
The CMLC were vitrified in closed straws as described in r WO2004 098285 and stored in liquid N2. A sterile filtered vitrification solution including vitro-PBS (Vitrolife AB) supplemented with 10% ethyleneglycol and 10% DMSO was used, as well as vitro-PBS (Vitrolife AB) supplemented with 0.3 M trehalose, 20% ethyleneglycol and 20% DMSO. The cells were recovered after thawing in culture medium (Knock Out DMEM supplemented with 20% FBS, 1% penicillin-streptomycin, 1% Glutamax, 0.5 mmol/l β-mercaptoethanol, and 1% non-essential amino acids).
In a specific embodiment the CMLC normally has a size of from about 20 μm to about 30 μm. Moreover, 50-80% of the cells in the cluster express cardiac markers.
A suitable product for shipping contains one or more clusters in KnockOut DMEM medium supplemented with 2-20% FBS, 1% penicillin-streptomycin, 1% Glutamax, 0.5 mmol/l β-mercaptoethanol and 1% non-essential amino acids (all from Invitrogen, Carlsbad, Calif.).
A composition according to the present invention provides a composition wherein the cluster retains at least 95% of its described characteristics after storage at a temperature of at least −80° C. for at least 2 years. Further, at least about 50% or more such as, e.g., about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more or about 95% or more of the cells are viable after storage of the composition at a temperature of at least −80° C. for at least 2 years.
Kit
The present invention further provides a kit for use in testing of cardiotoxicity of a specific substance, wherein the kit comprising
i) one or more cluster of cardiomyocyte-like cells or a composition as described above, and
ii) specific instructions for use of the cluster or composition, whichever is relevant.
In a specific embodiment the kit may comprise a composition of cardiomyocyte-like clusters of nodal-like cells and/or atrial-like cells and/or ventricle-like cells as defined above.
The kit may further comprise
iii) a medium into which the specific substance is dispersed before use of the kit.
In a further embodiment, the present invention provides a kit for use in in vitro testing during drug discovery of a specific substance, wherein the kit comprises
i) one or more clusters of cardiomyocyte-like cells or a composition as described above,
ii) specific instructions for use of the cluster or composition, whichever is relevant.
In a specific embodiment the kit may comprise a composition of cardiomyocyte-like clusters of nodal-like cells and/or atrial-like cells and/or ventricle-like cells as defined above.
The kit may further comprise
iii) a medium into which the specific substance is dispersed before use of the kit.
In a still further embodiment the present invention also provides a kit for regenerative medicine comprising
In a specific embodiment the kit may comprise a composition of cardiomyocyte-like clusters of nodal-like cells and/or atrial-like cells and/or ventricle-like cells as defined above.
The cryopreservation of the cardiomyocyte-like cell clusters may be performed by using a slow freezing method or by vitrification, using open or closed pulled straws.
Vitrification Solutions
At least one of the vitrification solutions (the first and the second solution) may contain one or more cryoprotectants or mixtures of cryoprotectants. Non-toxic cryoprotectants are of course preferable. Cryoprotectants help minimizing shrinking by reducing the mole fraction of other solutes remaining in the non-frozen water. They inhibit the formation of crystalline ice, and thus depress the freezing point of the water. They may also prevent protein denaturation by hydrogen binding with bound water. As cells cool, solvent water converts to extracellular ice, and the increasing extracellular concentration of non-permeating electrolyte or non-electrolytes damages the cells. When treated with a cryoprotectant, cells do not reach the salt concentrations of non-treated cells until they reach much lower temperatures. Chemical reactions proceed very slowly at such low temperatures and consequently cellular damage is minimized. Usually it is better to use a combination of cryoprotectants since there might be differences between different types. The cryoprotectants may also function as osmotically active agents. Suitable cryoprotectants can be selected from the group consisting of ethylene glycol, propylene glycol, dimethylsulfoxide, glycerol, propanediol, sugars including sucrose, trehalose, maltose, lactose and methyl pentanediol.
A vitrification procedure may comprise the following steps,
i) transfer of the cardiomyocyte-like clusters to a first solution,
ii) optionally incubation of cardiomyocyte-like clusters in the first solution,
iii) transfer the cardiomyocyte-like clusters obtained in step i) or ii) to a second solution,
iv) optionally incubation of the cardiomyocyte-like clusters in the second solution,
v) transfer of the cells obtained from step iii) or iv) into one or more, open, closed at least on one side or fully closed straws, and
vi) vitrification of the one or more open, closed at least on one side, or fully closed straws.
The concentration of the individual agents contained in the first and or the second solution is normally in a range of 5-50% v/v such as, e.g. from about 5% to about 40% v/v such as e.g. from about 5% to about 25% v/v (first solution) and from about 5% to about 30% v/v (second solution). Normally, the total concentration (i.e. calculated as v/v, w/v or M) of the cryoprotectant in the second solution is larger than that in the first solution. The first and the second solution may contain one or more cryoprotectants that are the same or different. The concentration of the one or more cryoprotectants in the first and the second solution can be the same or different, and normally the total concentration of the cryoprotectant in the second solution is larger than that in the first solution.
In a specific embodiment of the invention, the cryoprotectant is trehalose. The concentration of trehalose contained in the first and/or the second solution is normally within a range from about 0.02 M to about 1 M, such as, e.g., from about 0.05 M to about 0.9 M, from about 0.1 M to about 0.8 M, from about 0.2 M to about 0.7 M, from about 0.3 M to about 0.65 M, from about 0.4 M to about 0.6 M, from about 0.45 M to about 0.55 M. Usually, sucrose is used in similar applications. Trehalose is a unique, naturally occurring disaccharide and is found in hundreds of plants and animals. Trehalose is an important source of energy and has been shown to be a primary factor in stabilization of organisms during time of freezing. It has been shown that trehalose can depress the phase transition temperature of membranes so that they remain in the liquid-crystal state even when dry. Without being bound to any theory, it is hypothesized that this prevents membrane leakage during rehydration, thereby preserving cellular viability. With respect to proteins, trehalose has been shown to inhibit protein denaturation by exclusion of water from the protein surface when the cells are in the hydrated state.
In another embodiment of the invention, the cryoprotectant is sucrose. The concentration of sucrose contained in the first and/or the second solution is normally within a range from about 0.02 M to about 1 M, such as, e.g., from about 0.05 M to about 0.9 M, from about 0.1 M to about 0.8 M, from about 0.2 M to about 0.7 M, from about 0.3 M to about 0.65 M, from about 0.4 M to about 0.6 M, from about 0.45 M to about 0.55 M.
In yet another embodiment of the invention, at least one of the first and second solutions comprises a cryoprotectant.
At least one of the first and the second solution may comprise a viscosity-adjusting agent. Suitable viscosity-adjusting agent for use in the present context may be selected from the group consisting of Ficoll, Percoll, hyaluronic acid, albumin, polyvinyl pyrrolidone, alginic acid, gelatin and glycerol. The first and the second solution may contain one or more viscosity-adjusting agents that are the same or different. The concentration of the one or more viscosity-adjusting agents in the first and the second solution may be the same or different.
In a specific embodiment of the invention, the viscosity-adjusting agent is Ficoll. The concentration of Ficoll contained in the first and/or the second solution is at the most about 150 mg/ml, such as, e.g., at the most about 100 mg/ml, at the most about 50 mg/ml, at the most about 25 mg/ml, at the most about 15 mg/ml or at the most about 10 mg/ml.
In one embodiment of the invention, at least one of the first and second solutions is an aqueous solution.
In one embodiment of the invention, xenofree hBS cell lines and culture methods are applied for therapeutic applications of the cardiomyocyte-like cell clusters.
In one embodiment of the invention, the hBS cell line constitutes a trisomic cell line such as SA002 carrying an extra chromosome 13. This cell line has shown a great tendency for efficient development into cardiomyocyte like cell clusters, as well as a high yield of clusters. One advantage of developing CMLC using hBS line SA002 is that the cells carry an intrinsic “label” (i.e., three copies of chromosome 13) which can be readily detected (using standard genomic techniques) in mixtures of non-trisomic cells derived or isolated from other sources.
Improved Yield of CMLC from hBS Cells
Another aspect the present invention relates to a method for preparing CMLCs from hBS cells. The novel method has shown to be advantageous compared with prior art method using EB formation as the yield of beating clusters are markedly improved. The new method is also more suitable for large scale production. The cell lines used in this method may be obtained from a commercial available cell line, such as e.g. SA 002, SA 121, SA 001, SA002.5, and SA 461, available from Cellartis AB, Goteborg, Sweden.
Thus, the present invention provides a method for the preparation of a cluster comprising cardiomyocyte-like cells, the method comprising the steps of:
i) suspending and dissociating undifferentiated hBS cells in a culture medium,
ii) subjecting the thus dissociated aggregates to forced aggregation,
iv) incubating the thus forced aggregated cell aggregates in culture medium optionally comprising one of more growth factors to obtain one or more 3D structures,
v) transferring one or more 3D structures to one or more plates and incubating the 3D structures in culture medium optionally comprising one or more growth factors to develop them into one or more clusters comprising contracting cells.
The method may also comprise a further step of isolating the one or more clusters by removing the one or more clusters from said plate.
In one embodiment, the method for obtaining cardiomyocyte-like clusters, comprises the steps of
In the event that these cell lines are propagated on feeder layers, the cells may be detached from the feeder layer by use of mechanical or, preferably, enzymatic treatment, whereby a suspension of primarily undifferentiated hBS cells is obtained. The enzymatic treatment may be performed by use of a proteolytic enzyme, such as a collagenase, for an appropriate time, e.g. 2-20 minutes, 5-15 minutes or 10-15 minutes. However, it is envisaged that any treatment of the cells to obtain a cell suspension can be used in the present invention.
It is envisaged that the cells obtained in the above-mentioned step 1 are mainly undifferentiated, meaning that from about 70% or more, such as e.g. 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more of the cells are undifferentiated.
The cell suspension may thereafter be resuspended in an appropriate media which supports the cells, this media can for example be Knock Out DMEM supplemented with FBS (preferably in the range 15-25%, such as 20%). The culture medium can, at various time points during the experiments, be supplemented with different factors such as protein growth factors, chemical compounds, minerals, or other signalling molecules. Examples of such factors are BMP-2, BMP-4, BMP-5, TGF-β1, Activin A, Growth hormone, LIF, and PDGF. It is important to note that the suspension step is relatively short and does not allow for embryoid bodies to develop. In the present method the suspension step is just a transfer step. Optionally, the resuspended cells may thereafter be further dissociated into small aggregates of undifferentiated cells, in the event that the cells form large aggregates (e.g. above 0.5 mm). This dissociation can be performed manually, e.g. by using a pipette, until the cells forms small aggregates of the size 0.2-0.4 mm.
By thereafter transferring said cells to appropriate culture dish(es) in an appropriate medium (e.g. the same as mention above, supplemented with different factors), and incubating the cells for 1-30 days, e.g. 1-15 days, 1-10 days, 1-5 days, or preferably for 3 days, spontaneous clusters of contracting cardiomyocyte-like clusters appeared. The culture dish(es) used may be of any kind that supports the cells, but in a preferred embodiment the culture dish(es) are gelatine coated dishes.
The cell aggregates obtained in step 2) or 3) are subjected to forced aggregation and/or sedimentation before transferring the cells to the culture dish(es) in step 4) and 5).
By using this method 3D structures are formed when spun down through forced aggregation. The 3D structures are different from embryoid bodies. The advantage is that a more reliant structure is formed when compared to e.g. embryoid bodies, which have a tendency to fall apart, and therefore the present method gives a higher yield between the number of undifferentiated cells used and the resulting number of cardiomyocyte-like clusters. The method is also advantageous in that it is faster, more reliable and more scalable and useable for automation compared to the prior art EB protocols.
The preferred method for obtaining cardiomyocyte-like clusters, comprises the steps of
By using forced aggregation approximately 4-6 colonies from step 2) or 3) are transferred to a plate or tube appropriate for centrifugation, e.g. a 96-well v-bottom plate (Corning Incoporated, NY, USA). The cells are then centrifuged for 2-20 minutes at 100-800×g (e.g. 200-600×g for 5-10 min or, 300-500×g for 5-10 min, or at 400×g for 5 min.). These centrifuged cells may, before the transfer mentioned step 6), further be incubated for 1-10 days (such as 1-7 days, or 1-5 days) for the formation of 3D structures, which structures then are transferred according to step 6) above.
The forced aggregation may additionally be combined, or replaced, by sedimentation of the cell suspension obtained step 2) or 3), for 1-36 hours (such as 2-24 hours or 3-12 hours), before proceeding to step 6).
As mentioned above the yield of CMLC from hBS cells can be further improved by supplementing the culture medium used with different factors such as protein growth factors, chemical compounds, minerals, or other signalling molecules. In particular the inventors have found that when the culture medium (e.g. DMEM) comprises a member from the transforming growth factor beta superfamily (e.g. Activin A) and/or a member from the fibroblast growth factor family (e.g. bFGF) in e.g. step 5 and/or step 7 of the preferred method (and/or step 2 of the first embodiment of the method) the yield increases. This yield as shown in
The concentrations of Activin A supplemented to the culture medium comprises from about 5-40 ng/ml, such as e.g. 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, or 12 ng/ml; and the concentration of bFGF supplemented to the culture medium comprises from about 5-40 ng/ml, such as e.g. 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml or 14 ng/ml.
It has also been shown that a concentration of about 20% FBS (e.g. 15-25%) is advantageously added in step 5 and/or 7 of the preferred method to improve the yield.
In a specific embodiment the method for obtaining cardiomyocyte-like clusters, thus comprises the steps of
As mentioned the invention may also take place by sedimenting the cells instead of centrifugation. Specific embodiments of the invention thus comprises,
The yield have also been shown to increase (as shown in e.g.
The concentration of the GSK-3 inhibitor supplemented to the culture medium may be in the range of from about 1-25 μM, such as e.g. 2-10 μM or 5 μM, and the concentration of the p38 MAP-kinase inhibitor supplemented to the culture medium may be in the range of about 1-25 μM, such as e.g. 2-10 μM or 5 μM.
Further specific embodiments of the invention thus comprises,
The inventors have also shown that by supplementing the medium in step 7 of the preferred method (or the corresponding step 5 of the first mentioned embodiment of the method) containing Activin A and/or bFGF with LIF, the yield improved by approximately a 2 fold compared to controls that only receive medium, as shown in e.g.
Further specific embodiments of the invention thus comprises,
As mentioned hereinbefore, a cluster according to the invention can be used in in vitro studies (e.g. in drug discovery, drug testing, target identification and target validation, e.g. based on electrophysiological measurement on funny channels as described Examples 11 and 14.
The invention is further illustrated in the following non-limiting examples.
Spontaneously contracting cells were derived from undifferentiated hBS cells cultured on MEF cells (Heins et al 2004 Stem Cells). The cell lines used for this experiment could be the hBS cell line SA002, SA121, SA001, SA002.5, SA461 (Cellartis AB, Goteborg, Sweden, http://www.cellartis.com) and they can be propagated as described Heins et al. 2004. These cell lines are listed in the NIH stem cell registry and the UK Stem Cell bank. The hBS cells were detached from the feeder layer by incubation with collagenase IV (200 U/ml), for 10-15 minutes at 37° C. The cell suspension was transferred to a 15 ml tube, and after the cells had sedimented, the supernatant was removed. The colonies were resuspended in Knock Out DMEM supplemented with 20% FBS, 1% penicillin-streptomycin, 1% Glutamax, 0.5 mmol/l β-mercaptoethanol and 1% non-essential amino acids (all from Invitrogen, Carlsbad, Calif.) and dissociated mechanically into small aggregates of undifferentiated cells (0.2-0.4 mm) using a pipette. This cell suspension was distributed (200 μl/well) into a 96-well v-bottom plate (Corning Incorporated, NY, USA) at a concentration of approximately 4-6 colonies per well and centrifuged for 5 min at 400×g. The plate was then incubated at 37° C. for 3-8 days. The 3D structures that formed were then transferred to gelatine coated dishes containing culture medium (same as above). After a couple of days (normally 1-4) of culture, spontaneously contracting areas with cluster of contracting cells (CMLC) occurred and could be visually identified using a light microscope. Medium was changed every second to third day. New spontaneously beating areas continued to appear for up to 20 days post-plating. The clusters are isolated by mechanical dissection under visual inspection using a light microscope from the 3D-structures and subjected to further characterization.
The distribution in beating frequency was evaluated by determining the beats per minute (bpm) of 109 different spontaneously contracting areas. The average beating frequency was 44 bpm±24 (Std.dev.). The overall distribution is illustrated in
In order to improve the yield of CMLC from hBS cells, the culture medium can, at various time points during the experiments, be supplemented with different factors such as protein growth factors, chemical compounds, minerals, or other signalling molecules. Examples of such factors are BMP-2, BMP-4, BMP-5, TGF-β1, Activin A, bFGF, Growth hormone, LIF, and PDGF.
Cell Culture and Differentiation
The hBS cell line SA002 (Cellartis AB, Goteborg, Sweden, http://www.cellartis.com) was propagated as described Heins et al. 2004. This cell line is listed in the NIH stem cell registry and the UK Stem Cell bank. Differentiation of hBS cells was performed as described in Example 1 above. Using light microscopy clusters of spontaneously contracting cardiomyocyte-like cell clusters (CMLC) are frequently observed when hBS cells are differentiated through this protocol. For microarray experiments, three separate samples (biological replicates) of CMLC were collected. For comparison purposes, two independent samples from undifferentiated hBS cells and one sample from a mixed population of differentiated cells (i.e., no contracting CMLC present) were also collected for the analysis. The mixed population was collected as the remaining population of differentiated cells following the selection and removal of the spontaneously contracting CMLCs in the cultures differentiated as described in Example 1. Samples from three independent runs were pooled as indicated below. Using the mixed population as a control therefore represents a differentiated cell population, minus the CMLCs, making subtraction of genes up regulated in all differentiated cells available.
Samples: 1. Undifferentiated hBS cells (pooled from passage 33, 36, 37, 39, and 40)
RNA Extraction and Micro Array Experiments
Total RNA was extracted from undifferentiated and differentiated hBS cells using the RNeasy® Mini Kit (Qiagen) and subsequently used for microarray experiments employing GeneChip® Human Genome U133 Plus 2.0 (Affymetrix Inc, Santa Clara, Calif., USA) targeting 54,675 transcripts. Due to small amounts of RNA, some of the samples in the experiment were amplified using two-cycle in-vitro transcription (IVT). RNA quality and concentration was measured using an Agilent 2100 bioanalyzer and Nanodrop ND-1000 respectively. Total RNA was processed following the GeneChip® Expression 3′-Amplification Reagents Two-cycle cDNA synthesis kit** instructions (Affymetrix Inc, Santa Clara, Calif., USA) to produce double-stranded cDNA. This was used as a template to generate biotin-targeted cRNA following manufacturer's specifications. Fifteen micrograms of the biotin-labelled cRNA was fragmented to strands between 35 and 200 bases in length, 10 micrograms of which was hybridised onto the Gene Chip® Human Genome U133 Plus 2.0 (Affymetrix Inc, Santa Clara, Calif., USA) overnight in the GeneChip® Hybridisation oven 6400 using standard procedures. The arrays were washed and then stained in a GeneChip® Fluidics Station 450. Scanning was carried out with the GeneChip® Scanner 3000 and image analysis was performed using GeneChip® Operating Software. SCIBLU—Swegene Centre for Integrative Biology at Lund University (http://www.lth.se/sciblu) conducted the quality controls, the RNA processing and the hybridization of the arrays. The samples representing undifferentiated hBS cells, differentiated contracting CMLC and a mixed population of differentiated cells were hybridized to the arrays. The arrays were run in duplicates.
Data Analysis
In order to define the family of genes that are up-regulated in the CMLC the fold change (FC) in gene expression between undifferentiated hBS cells and the three different biological replicates of CMLC was calculated. Notably, in these calculations the amplified and the un-amplified RNA samples were kept separate. Genes that were called as Absent by the MAS software in all samples were filtered and not included in further analyses. In the amplified samples 17,436 probes were called as Absent in all samples and in the un-amplified samples 18,534 probes were called as Absent in all samples and these genes were filtered from the dataset. When the FC-values had been calculated for each of the CMLC samples, the average of the FC-values (both amplified and un-amplified FC-values) were determined and subsequently used when filtering the genes. Genes with an average FC-value<2 across all CMLC samples when compared to undifferentiated hBS cell samples were filtered from the data set and only the remaining 9,801 genes were considered for further analysis. Table I illustrates the distribution of up-regulated genes and summarizes the number of genes at specific FC levels (2, 3, 5, and 10).
To further refine the selection method, a population of “mixed differentiated hBS cells” was included as control material to distinguish genes up regulated in all types of differentiated cells from genes specifically up regulated in cardiomyocyte-like cell clusters. The “mixed differentiated hBS cells” thus represent a population of spontaneously differentiated hBS cells where cells from all three germ layers mesoderm, endoderm and ectoderm may be present.
To identify genes that were specifically up-regulated in CMLC and not in the mixed differentiated hBS cell population, the following parameters were calculated:
For all calculations, the average of the three replicates of CMLC was used. The following criteria were used to sort out only genes that were specifically up-regulated in the CMLC:
The remaining 56 genes are listed in Table II and these represent suitable marker genes for the CMLC.
Of particular note is that the majority of the genes in Table II can be classified into two different groups. The first group consists of genes previously associated with cardiac cells and the second of genes previously associated with endodermal cells, such as hepatocytes. Examples of genes belonging to the first group are (marked in light grey in table II): MYH6, MYL7, MYL4, TNNC1, TNNT2, PLN, UN, MYH7, LDB3, NPPB, GATA6, MYL3, CSRP3, ACTN2, MB, MYOZ2, TBX5, and HSP27. Examples of genes belonging to the second group are (marked in dark grey in table II): AFP, TF, APOA2, AHSG, SERPINA1, APOA1, ALB, APOC3, TTR, APOB, and RBP4.
To get an even stricter selection of genes specifically up-regulated in CMLC the criteria were narrowed to:
The remaining 9 genes are listed in Table III and these represent suitable marker genes for the CMLC.
The selection shows an even distribution between endodermal and mesodermal genes expressed in the clusters. The mesodermal genes and the endodermal are marked in, light grey and dark grey respectively in Table III.
Microarray Analysis of Cardiomyocyte-Like Clusters Using Significance Analysis of Microarrays (SAM) Algorithm
By using the differentiation method and RNA extraction method described above, RNA were pooled at various time points (<22 days) after the onset of contraction.
Undifferentiated hBS were harvested at day 4-5 after passage for RNA extraction or differentiated to obtain CMLC using cells in passage 23-41. Spontaneously contracting clusters were identified by visual inspection using light microscopy and harvested by mechanical dissection. Specific care was taken only to harvest the beating areas with a minimum of surrounding non-contracting cells.
For each experiment, the material consisted of one pooled sample of undifferentiated hBS and two different biological replicates of pooled clusters.
Data Analysis
Identification of Differentially Expressed Genes
Genes that were significantly up- or down-regulated in the hBS-derived clusters compared to undifferentiated hBS were identified using the SAM algorithm (Tusher V G, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA. 2001;98:5116-5121). Briefly, the algorithm assigns a score to each gene based on differences in expression between conditions relative to the standard deviation of repeated measurements. The false discovery rate (FDR) is determined by using permutations of the repeated measurements to estimate the percentage of genes identified by chance. The algorithm was applied to each of the data sets from the two experiments separately using FDR<0.04. Subsequently, only the genes marked as significantly up- or down-regulated in both data sets were considered as differentially expressed in CMLC compared to undifferentiated hBSs.
Results
Using the SAM algorithm, and FDR<0.04, 530 genes were identified that were significantly up-regulated in clusters compared to undifferentiated hBS,
In addition, we also identified a smaller group of 40 genes that were significantly down-regulated in the hBSC-derived CMLC compared to undifferentiated hBSC,
To characterize the family of up-regulated genes in hBSC-derived CMLCs, we performed a Gene Ontology analysis (GO) analysis. Gene Ontology (GO) annotations were used to group the genes according to biological process, molecular function, and cellular component. By comparing with a reference list, overrepresentation of annotations among sets of genes can be calculated as O/E where O is the observed number of genes with a specific annotation and E is the expected number of genes with that annotation. E is calculated as E=R*I/R where R is the number of reference genes and I is the number of interesting genes. All genes represented on the arrays were used as the reference list. Significantly overrepresented annotations (p<0.01) among the up-regulated genes from all three annotation categories at level 5 in the GO annotation database were identified using the hypergeometric test.
Notably, several of the significantly overrepresented GO annotation terms shown in
To investigate the possible interactions among proteins from the significantly up-regulated genes in CMLC, the search tool STRING (http://string.embl.de/) was used to mine for recurring instances of neighbouring genes (Snel B, Lehmann G, Bork P, et al. STRING: a webserver to retrieve and display the repeatedly occurring neighbourhood of a gene. Nucleic Acids Res. 2000;28:3442-3444. STRING aims to collect, predict and unify various types of protein-protein associations, including direct (physical) and indirect (functional) associations. Using the list of up-regulated genes in CMLC as input to STRING, 431 matches were made and potential interactions among products from these genes were investigated further. A protein interaction map was created from these gene products and compared with protein interaction maps from 10 different randomly generated sets of genes, all of equal size. For each protein, an interaction score was calculated as n*2/N, where n is the number of interactions (edges in the map) for the protein in question and N is the total number of input proteins. Furthermore, the number of hub proteins was determined. Following Han et al., we designated a protein as a hub if it had ≧5 interactions with other proteins (Han J D, Bertin N, Hao T, et al. Evidence for dynamically organized modularity in the yeast protein-protein interaction network. Nature. 2004;430:88-93. A distinction between “party hubs”, which interact with most of their partners simultaneously, and “date hubs”, which interact with their partners at different times or locations has also been made. Since our data sets do not include time series data we identified only “party hubs”. As default STRING utilizes four different sources; Genomic context, High-throughput experiments, Co-expression, and Previous knowledge, to derive protein interaction maps. However, we restricted our analysis to include only experimentally determined protein interactions, excluding for example text mining, to increase the validity of the results.
As shown in
In addition to identifying protein interaction networks among the differentially expressed genes we also here report significant up-regulation of a number of cellular pathways. Strikingly, 23 genes in the Focal adhesion pathway are significantly up-regulated in hBSC-derived CMLCs. The Focal adhesion pathway has been implicated in a diverse array of cellular processes, including tissue remodeling, cell migration, embryogenesis, growth factor signaling, cell cycle progression, and cell survival (Parsons et al 2003, Petit et al 2000). In addition, the role of focal adhesions in mechanotransduction in cardiomyocytes has recently been highlighted (Samarel et al 2005). Interestingly, besides affecting the beat-to-beat regulation of cardiac performance, mechanotransduction also influences the proliferation, differentiation, growth, and survival of the cellular components that comprise the human myocardium. Furthermore, in neonatal rat ventricular myocytes it has been reported that focal adhesion kinase regulates the activation of the MEF2 and JNK/c-Jun pathways, which have important roles in the early activation of the hypertrophic genetic program by mechanical stress in cardiomyocytes (Nadruz et al 2005).
Notably, 16 genes in the Calcium signaling pathway are also significantly up-regulated in the hBSC-derived CMLCs. This observation is not unexpected since Ca2+, as an intracellular messenger, is an important component for the initiation and regulation of cardiac contraction (Ferrier G R, Howlett S E. Cardiac excitation-contraction coupling: role of membrane potential in regulation of contraction. Am J Physiol Heart Circ Physiol. 2001;280:H1928-1944.). In addition, Ca2+ has been shown to be important already at the beginning of life to mediate the process of fertilization and later on it regulates some of the cell cycle events during early development (Berridge et al 1997). In this regard, the Hedgehog signaling pathway, also known to be critical in a plethora of developmental processes (Ingham et al 2006) is significantly up-regulated in the hBSC-derived CMLCs. Specifically, Sonic hedgehog appears to be a critical signaling factor for cardiac development in P19 cells (Gianakopoulos et al 2005). In addition, experimental mice lacking hedgehog signaling show a delay in the expression of Nkx2.5 (Gianakopoulos et al 2005).
Cell Culture and RNA Extraction
Spontaneously contracting CMLC were derived from undifferentiated hBS cells (SA002) (see Example 1). The hBS were detached from the MEF feeder layer by incubation with collagenase IV (200 U/ml), for 10-15 minutes at 37° C. The suspension was transferred to a 15 ml tube, and after the cells had sedimented, the supernatant was removed. The colonies were re-suspended in culture medium and dissociated mechanically into small aggregates of undifferentiated cells using a pipette. This cell suspension was distributed (200 μl/well) into a 96 well plate (Costar, Corning, untreated, v-bottom) and centrifuged for 5 min at 400×g. The plate was then incubated at 37° C. for 6-8 days. The 3D structures that formed were then transferred to gelatine coated dishes containing culture medium. After a couple of days spontaneously contracting clusters of cells (CMLC) were observed. Some of these clusters were harvested after 0-2 weeks (“early CMLC”) after onset of contraction while others were maintained in culture for at least 6 weeks (“late CMLC”) before harvest. In parallel, undifferentiated hBS cells were harvested for subsequent RNA extraction.
Samples: 1. Undifferentiated hBS (passage 35-37)
Total RNA was extracted from undifferentiated hBS cells and CMLC using the RNeasy® Mini Kit with on-column DNase treatment (Qiagen).
Quantitative Real-Time PCR
To investigate the gene expression level of specific ion-channels in the CMLC and undifferentiated hBS cells, TaqMan® low density arrays (384-Well Micro Fluidic Cards, Applied Biosystems) were used. For comparison, commercially available total RNA from human adult normal heart tissue (pool from 6 different male donors) (BioChain®) was analyzed in parallel. cDNA synthesis was performed using Superscript 3 (cat no 18080-051 Invitrogen). Each PCR reaction was run in quadruplicate. The relative gene expression levels were normalized against the expression of GAPDH and calculated according to the ΔΔACt-method. The following genes were analyzed: POU5F1 (Oct-4), NANOG, NKX2.5, GATA4, TNNI3, DES, SCN5A, SCN1B, SCN2B, CACNA1C, CACNB1, CACNB2, CACNA2D1, CACNA1H, KCNQ1, KCNE1, KCNH2, KCNE2, KCNA5, KCNAB1, KCNAB2, KCNJ2, KCNJ12, KCNJ3, KCNJ5, KCNJ11, ABCC9, KCND3, KCNA4, Kcnip2, HCN4, KCNK1, CFTR, GJA5, GJA1, GJA7, ATP2A2, SLC8A1, RYR2, FKBP1B, SLC9A1, MYL2, MYLK, MYH6, and MYH7. The results from the PCR analysis are summarized in
Cardiomyocyte-like cell clusters (CMLC) were derived from undifferentiated hBS cells as described in Example 1. The clusters of spontaneously contracting cells were manually dissected and further divided into smaller clusters (about 0.2-0.3 mm in diameter). The CMLC were vitrified in closed straws as described in Patent application “Vitrification” publication number WO2004 098285 and stored in liquid N2. A sterile filtered vitrification solution including vitro-PBS (Vitrolife AB) supplemented with 10% ethyleneglycol and 10% DMSO was used, as well as vitro-PBS (Vitrolife AB) supplemented with 0.3 M trehalose, 20% ethyleneglycol and 20% DMSO. The cells were recovered after thawing in culture medium (Knock Out DMEM supplemented with 20% FBS, 1% penicillin-streptomycin, 1% Glutamax, 0.5 mmol/l β-mercaptoethanol, and 1% non-essential amino acids) and the results summarized below.
Experiment 1: 10 CMLC were vitrified
Experiment 2: 12 CMLC were vitrified
Experiment 3: The results are summarized in Table IV below.
Additional experiments were also performed and are summarized below. The table below reports the collected data from four separate vitrification experiments. This data shows the percentage of cardiomyocyte clusters that are attached and those that are attached and spontaneously contracting at three, seven or 14 days after thawing.
Colonies of undifferentiated hBS cells are separated from their feeder cells, using collagenase (200 U/ml, in DMEM/F12 medium). Colonies are collected either through passive sedimentation or via centrifugation. The colonies are then slightly broken up by gentle pipetting, and resuspended to give a final concentration of 4 average-sized colonies per 200 μl medium (Medium 1). Colonies are then centrifuged in a 96 well, v-bottom, uncoated plate (Corning article number: 3896) for 5 minutes at 400×g, with 200 μl (4 colonies) per well. The plates are then stored in an humidified incubator at 37° C., with 5% CO2 to allow 3D structures containing differentiated cells to form over a 3-8 day period. After this period, the 3D structures are transferred to gelatine coated Petri dishes and cultured for a period of two weeks in medium 2, below, with medium changes every two/three days.
Medium 1 for 3D structure formation:
39.4 ml DMEM with glutamine and 1% penicillin/streptomycin
500 μl non essential amino acid
100 μl β-mercapoethanol
10 ml foetal bovine serum
Medium 2 for plating and culturing of 3D structures to allow for spontaneous differentiation: as above, but with either 20% or 2% FBS to give a better differentiation.
Cluster Characteristics
When 3D structures are plated at age 7 days, on gelatine in medium containing 20% foetal bovine serum, clusters with contracting cells (CMLC) have the following characteristics (n=55):
The distribution of beating frequency is affected by both the plating age of the 3D structures from which the cardiomyocytes-like cell clusters are obtained, and the culturing conditions after the plating of the 3D structures.
Cardiomyocyte-like cell clusters (CMLC) were derived from undifferentiated hBS cells as described in Example 1. The clusters of spontaneously contracting cells were manually dissected and further divided into smaller clusters (about 0.2-0.3 mm in diameter). In some experiments, the cell clusters were cryopreserved and in others the clusters were dissociated into single cell suspensions using trypsin/EDTA (15 min at 37° C.). The cell suspensions or cell clusters were transferred to cryopreservation media. The following different media compositions have been tested with comparable results regarding recovery of viable cells post-thawing:
1. (KO-DMEM, NEAA, β-MeOH, Glutamax, PEST, 20% FBS)+10% DMSO
2. 90% FBS+10% DMSO
3. 95% FBS+5% DMSO
4. 45% KO-DMEM+45% FBS+10% DMSO
5. 50% KO-DMEM+45% FBS+5% DMSO
6. 50% Trypsin/EDTA+40% FBS+10% DMSO
7. 20% KO-DMEM+50% Trypsin/EDTA+20% FBS+10% DMSO
Cardiomyocyte-like cell clusters (CMLC) were derived from undifferentiated hBS cells as described in Example 1 and cryopreserved as described in Example 4. The CMLC were thawed and pharmacological test were performed using different platforms, such as micro-electrode arrays, voltage-clamp analysis, transmembrane action potential (TAP) measurements, and QT-screen system. The use of the functional clusters is further illustrated in
Spontaneously beating CMLC were derived and isolated from hBS cells as described in Example 1. The CMLC were plated on microelectrode arrays (MEA). After adhesion of the CMLC to the MEA surface, the electrical activity was recorded as extracellular field potential by the substrate integrated electrodes of the MEA. Cardiac field potential properties have been studied before and their correlation with cardiac action potential data has been well described (Banach et al Am. J. Physiol. 2003, 284 (6): H2114-H2123). The addition of the QT-prolonging drug E-4031 causing hERG channel blockade was measured as a delayed repolarisation of the cardiac field potentials. Increasing drug concentrations (0, 100 pM, 1 nM, 10 nM, 100 nM, 1000 nM) was added to obtain cumulative dose response curves. Notably E-4031 did not affect beating frequency over the experiments but caused a sustained prolongation in the low nanomolar concentrations of the cardiac field potential. This is indicative for a hERG channel block known to be caused by this compound. This example is illustrated in
In addition, similar experiments were performed using other substances such as Astemizol (antihistaminic) and Dofetilide (a class III anti arrhythmic drug). As shown in
CMLC were derived from undifferentiated hBS cells (cell line SA002, LOTAL002, passage 21, 22, 44 and 51) essentially as described in Example 1. In order to improve the yield of CMLC from hBS cells, the culture medium used before plating of the 3D structures (pre-plating) was supplemented with Activin A (10 ng/ml) and bFGF (12 ng/ml).
The number of contracting CMLC was visually determined at day 6 or 7 after plating of formed 3D structures. In three experiments, there was on average 4.7 times more beating CMLC in cultures that had been treated with Activin A and bFGF during the pre-plating period compared to control cultures not receiving the treatment (
Total RNA was isolated from mixed cultures, including CMLC, from the above mentioned experiments on day 7 after plating and transcribed into cDNA. Quantitative real-time PCR was performed using the Taqman labelling system and data were normalized to the endogenous control CREBBP. The expression of genes involved in early (Nkx2.5 and GATA4) and late (Troponin T2 and alpha-cardiac actin) cardiac differentiation was increased by adding Activin A and bFGF to the medium during the pre-plating period in combination with plating in CM medium supplemented with both 20% FBS (
By modifying the basic protocol described in Example 1 it is possible to increase the yield of CMLC. In this example some of these opportunities are illustrated.
The serum component used has an effect on the yield of beating CMLC from hBS cells. In a set of experiments different FBS preparations were used; standard FBS (as in Example 1) (Invitrogen, art nr 10108-165), dialyzed FBS (Invitrogen, art nr 26400-036), and charcoal stripped FBS (Invitrogen, art nr 12676-011). The cells were maintained in the presence of the different FBS (20%) during culture in the 96-well plates. The aggregates formed were plated after 3 days in new dishes coated with gelatine and subsequent culture was done in the presence of standard FBS (20%). The number of beating CMLC was determined 14 days after plating and the results are shown in
In addition to modulating the initial phase of differentiation (i.e., in the 96-well plates) it is also possible to stimulate later stages of differentiation to increase the yield of contracting CMLC from hBS cells. In this regard, hBS cells were differentiated in the absence or presence of Activin A and bFGF as described in Example 9. The differentiated cells (3D-aggregates) were plated at day 7 in medium supplemented with 1000 U/ml LIF. After 4 days the medium was changed to medium without LIF. The number of beating CMLC was counted at day 7 and day 14 after plating and the results are shown in
As an alternative to using protein growth factors for modulation of the differentiation of hBS cells it is possible to use low molecular weight compounds. Undifferentiated hBS cells were treated essentially as described in Example 9. In order to improve the yield of CMLC from hBS cells, the culture medium used before plating was supplemented with 5 μM SB 216763 (GSK-3 inhibitor) or 5 μM SKF-86002 (p38 MAP-kinase inhibitor) in addition to Activin A (10 ng/ml) and bFGF (12 ng/ml). The number of contracting CMLC was visually determined at day 7 after plating of formed 3D structures and the results are shown in
The day at which the 3D-aggregates are being plated after onset of differentiation also affect the yield of beating CMLC. In a series of experiments, 3D-aggreates of differentiating hBS cells were generated as described in Example 1. The 3D-aggregates were subsequently plated at day 7 (control) or earlier (day 3, 4, or 5) in new culture dishes. The number of spontaneously beating CMLC was visually determined using a light microscope and the data are summarized in
Methods
Transmembrane action potentials (TAP) were recorded by using a microelectrode filled with 3 M KCl (resistance 10 to 20 MΩ) connected via an Ag/AgCl junction to a high impedance amplifier (Intracellular Electrometer IE-210, Warner Instrument Corporation). Sharp microelectrodes were prepared using a glass, and a micromanipulator was used to lower the electrode into the bath and enter the cluster. The same impalement was kept for as long as possible. The action potentials were recorded on a PC (sampling frequency 1 and 20 kHz) and the different action potential characteristics were analyzed using custom-designed software (Pharm-Lab v6.0b4, AstraZeneca) to determine AP duration at 50%, 70% and 90% of repolarization (dur50, dur70, dur90), AP amplitude (amp), membrane resting potential (MRP), and maximum rate of rise of the AP upstroke (Vmax). Steady-state recordings of action potentials at spontaneous frequency or during electric stimulation of 1-4 Hz were made. All calculations were made on the average of 10 (consecutive) action potentials. The bath was continuously perfused with Tyrode's solution consisting of (in mmol/L) 130 NaCl, 4 KCl, 0.5 MgSO4.7H20, 1.8 NaH2PO4xH2O, 18 NaHCO3, 5.5 glucose, 1.8 CaCl2 and kept at 37° C.
CMLC were derived from hBS cells as described in Example 1 and 9 and the clusters were grown on gelatine coated silicon pieces and on cover glasses, which allowed for easy transfer into the bath, and eliminated the problem of keeping the cluster at the same position. Electrical stimulation of the CMLC was done using field stimulation by means of a mobile electrode.
0.5-1 μM E-4031 was used for testing the effect of blocking the rapid rectifier potassium current IKr. Change in APD was calculated and used to determine prolongation and assess triangularization.
To investigate the role of the funny current (If) in spontaneously contracting CMLC, 10 μM zatebradine was administered.
Results
The cells were characterized as either nodal-like, atrial-like, or ventricle-like based on the results of transmembrane action potential (TAP) recordings. The characteristics of action potentials recorded from beating CMLC are shown in
Characteristics of action potentials recorded from beating CMLC derived from hBS cells.
Although there is variability between individual CMLC, examples of close to fully mature ventricular APs were found. As an example,
Repeated recordings were made at different locations in the same beating CMLC cluster, showing a low variability within the clusters (
In the native heart, cardiomyocytes respond to increased beating frequency by shortening of APD. This rate adaptation is fundamental and can be impaired in some diseases. To test the ability of hBS-cell derived CMLC to respond equally to a change in beating frequency, clusters were paced by means of electrical field stimulation and APD90 was compared at different frequencies.
Spontaneous contractile activity in CMLC clusters is due to funny currents (If). If can be blocked by Zatebradine. Application of 10 μM of Zatebradine slowed beating frequency and increased duration as shown in
Change in % after administration of 10 μM of If blocker Zatebradine on atrial-like hESC-CM clusters (n=4).
The proarrhytmic potential was assayed using E-4031 which is a potent IKr blocker. E-4031 induced concentration dependent action potential prolongation in all CMLC phenotypes as summarized in the table below. It also caused triangularization as an effect of the drug, triggered activity, and in some cases EADs were detected (
Average change in percent of APD90±SD (n) for ventricular-, atrial-, and nodal-like CMLC APs
3D aggregate cultures were derived from undifferentiated hBS cells essentially as described in Example 1. The cell line used for derivation was SA002, LOTAL002 (passage 25, 62 and 68). In order to improve the yield of CMLC from hBS cells, the culture medium used before plating was supplemented with different combinations of 1-25 uM SB 216763 (GSK-3 inhibitor), 1-25 uM SKF-86002 (p38 MAP-kinase inhibitor) in addition to 1-20 ng/ml Activin A and 1.2-24 ng/ml bFGF. Three concentrations in each range were tested according to the scheme below. The number of contracting CMLC was visually determined (
The combination of factors that produced the highest number of beating areas related to the amount of start material was 20 ng/ml Activin A, 24 ng/ml bFGF, 25 μM SB 216763 and 1 μM SKF-86002 (group 4,
Spontaneously contracting CMLC were derived from undifferentiated hBS cells (SA002) essentially as described in Example 1. The hBS cells were detached from the MEF feeder layer by incubation with collagenase IV (200 U/ml), for 10-15 minutes at 37° C. The suspension was transferred to a 15 ml tube, and after the cells had sedimented, the supernatant was removed. The colonies were re-suspended in culture medium and dissociated mechanically into small aggregates of undifferentiated cells using a pipette. This cell suspension was distributed (200 μl/well) into a 96 well plate (Costar, Corning, untreated, v-bottom) and centrifuged for 5 min at 400×g. The plate was then incubated at 37° C. for 3-4 days. The 3D structures that formed were then transferred to gelatine coated dishes containing culture medium. After a couple of days spontaneously contracting clusters of cells (CMLC) were observed and 4 to 6 weeks thereafter all CMLC clusters were harvested for cryosectioning, i.e. they were manually isolated, washed two times in 1×DPBS (GIBCO) buffer, incubated for 10 minutes in 10% sucrose, embedded in Tissue-Tek (Histolab) and finally frozen in −80° C. Cryosections (18 μm) of 4-6 weeks CMLC clusters, as well as undifferentiated hBS cells on IVF plates, were fixed in 4% PFA and blocked with 1% BSA/0.01% Triton X-100 in 1×DPBS (GIBCO) buffer. The primary antibodies were diluted in blocking buffer and incubated over night at 4° C. Three 5 minutes washes in 1×DPBS buffer were followed by two hours incubation of diluted secondary antibody at room temperature.
Nuclei were visualized using blue-fluorescent 4′,6-diamindino-2-phenylindole (DAPI, 1:1000). After three times washes with DPBS, slides were mounted in DakoCytomation Fluorescent Mounting Medium (DAKO) and imaged with a Nikon E400 fluorescent microscope. Sections from at least 10-15 different CMLC clusters were analysed for each marker and the results were consistent across all the samples. All antibodies were diluted as fallows: rabbit monoclonal anti cardiac Troponin I (Abcam, ab52862) 1:250, mouse-IgG2b anti Oct-3/4 (Santa Cruz, SC-5279) 1:500, mouse-IgG3 anti SSEA-4 (Santa Cruz, SC-21704) 1:200, and mouse-IgM anti TRA-1-60 (Santa Cruz, SC-21705) 1:200. Secondary antibodies were diluted as fallows; donkey anti-rabbit AlexaFluor594 1:500 and donkey anti-mouse AlexaFluor488 1:500 both bought from Molecular Probe Inc (Oregon, USA).
In conclusion, no expression of the three well defined hBS cell markers (Oct-3/4, SSEA-4, and TRA-1-60) could be detected in hBS cell derived CMLC (
CMLC were derived from undifferentiated hBS cells essentially as described in Example 1 using cell line SA002. Zatebradine is a substance known to block so called funny channels, e.i. hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels 1-4, which are involved in the regulation of cardiomyocyte contraction and cardiac rate. In order to investigate the effect of Zatebradine on CMLC derived from hBS cells and to correlate this effect to age and gene expression, Zatebradine was administered at a concentration of 5 μM to 32 spontaneously beating clusters of different ages. The contraction rate was registered visually every other minute until the effect of the substance had declined. Gene expression was quantified using real-time RT quantitative PCR. The beating of some clusters was completely blocked by Zatebradine administration. On average, Zatebradine decreased the beating frequency of CMLC about 50% (
Cryosections of CMLC clusters were prepared and used for immunohistochemcial analysis as described in Example 13. Nuclei were visualized using blue-fluorescent 4′,6-diamindino-2-phenylindole (DAPI). The primary antibodies used were: rabbit polyclonal anti KCNH2 (Abcam, ab32585) and rabbit monoclonal anti cardiac Troponin I (Abcam, ab52862). The results are shown in
To test whether the cell clusters has functional receptors, the following method described in Norström et. al. 2006 can be used. The following pharmacological agents are used: noradrenaline and adrenaline (Apoteket, Umeå, Sweden); phenylephrine and forskolin (Sigma Chemicals, St. Louis, Mo., USA); phenoxybenzamine and acetylcholine (KeLab, Göteborg, Sweden); atenolol and atropin (NM Pharma AB, Täby, Sweden); labetalol (Glaxo Smith Kline, Mölndal, Sweden); verapamil (Abbot Scandinavia AB, Solna, Sweden).
Phenylephrine, an α1-adrenoceptor agonist, is administered to contracting areas at increasing concentrations in 30 min intervals. Contractile activity is stimulated in a dose-dependent manner (see Table 1 in Norström et al.). The stimulatory effect is more pronounced in specimens with low basal contraction frequency. It is noted how many beating areas that are non-responsive to phenylephrine. Phenylephrine (10-7 M), administered to arrested areas, initiates contractile activity promptly. The stimulatory effect of phenylephrine is counteracted by phenoxybenzamine, a blocker of α1-and α2-receptors. Irregular contractions can occasionally be observed at maximal stimulation by phenylephrine but normally returns to regular beats following inhibition by phenoxybenzamine. If primarily administrated to contracting clusters of cells, phenoxybenzamine, is expected to reduce the contraction frequency. This effect is immediate and may be followed by a successive restitution of contraction frequency. The inhibition can be reversed by phenylephrine.
Adrenaline, exhibiting predominantly β1-adrenoceptor agonistic effects in the myocardium, can be used to test stimulated contractile activity. The stimulatory effect is registered within minutes and maximal stimulation occurs at a certain. In specimens exhibiting low contraction frequency (<20 bpm) an increase of frequency may be observed at certain concentrations. The stimulatory effect is reduced within minutes following administration of atenolol, a β-receptor blocker. Likewise, labetolol, a β- and α1-receptor blocker can be used to show a reduction in the stimulation of contraction frequency exerted by adrenaline. Atenolol (10-7 M), may reduced spontaneous contractile activity within 2 min.
Noradrenaline, an α- and β1-adrenoceptor agonist, stimulates contractile activity normally in a concentration-response manner. Maximal stimulation is reached within 10 min and this response can be reversed by phenoxybenzamine. Noradrenaline (10-7 M) administered to an arrested cell can initiate contractile activity within minutes.
Acetylcholine, exerting muscarinic effect in the myocardium, reduces the contractile activity. Following administration of 10-4-10-3 M acetylcholine total inhibition may be observed and it may persist for hours. Atropin, which competes with acetylcholine for a common binding site on the muscarinic receptor, counteracts the inhibition by acetylcholine. Primarily administered atropin (10-6 M) stimulates spontaneous contractile activity. Verapamil, a blocker of the trans-membranous flow of Ca2+ ions and inhibitor of mobilization of intracellular Ca 2+, reduces or abolishes the contractile activity, depending on the concentration primarily administered. Spontaneous contractions of basal frequency can be reestablished following washing and exchange of medium within 30 min.
Forskolin is generally recognized as a stimulator of adenylate-cyclase and formation of cyclic adenosine monophosphate (cAMP). A stimulatory response may be registered after 30 min of treatment with 10-10-10-6 M of forskolin as compared with baseline frequency. Two beating areas are exposed to pico-molar concentrations of forskolin. In these areas the contractile activity may be inhibited at 10-12 M during registration between 5 and 30 min but stimulated at higher concentrations of forskolin, though an initial but transient inhibition may be observed 5 min after administration.
Based on the variation in spontaneous contraction frequency of cell clusters obtained from different EBs it is likely to assume that the cells represent atrial, ventricular as well as nodal cardiac muscle cells. Nevertheless, their response to chronotropic agents was principally similar although, generally, in cell clusters exhibiting low beat frequency, the response to adrenoceptor agonists was more pronounced. Thus, our results (Table 1) support the notion of the development of functioning both adrenergic and cholinergic mechanisms as an early event in embryonic cardiomyocyte differentiation as indicated previously (3, 4, 6). The endogenous spontaneous contractile activity and the response to pharmacological agents appeared unaffected by repeated experiments since the basal contractile activity returned after washing and the pharmacological response could be repeated after weeks in culture. Exceptionally, contracting clusters did not respond to any chronotropic agent which may imply an impediment of receptor differentiation. It has previously been shown that the response to adrenoceptor agonists is time-dependent and better recognized in more differentiated cardiomyocytes (4).
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Specific Embodiments are as Follows:
Item
1. A cluster comprising cardiomyocyte-like cells, the cluster comprising genes that are up-regulated and have,
i) expression values of 500 or more,
ii) a fold change in gene expression between cardiomyocyte-like cells and undifferentiated hBS cells (FCCMLC) of 10 or more.
2. A cluster according to items 1 and 2, the cluster comprising genes that are up-regulated further have,
iii) a ratio between FCCMLC and FCMC (i.e. the fold change between mixed differentiated hBS cells and undifferentiated hBS cells) of 10 or more.
3. A cluster according to any of the preceding items, derived from BS cells such as, e.g., hBS cells.
4. A cluster according to any of the preceding items, wherein the derived BS cells are trisomic hBS cells carrying an extra chromosome 13, such as SA002 carrying an extra chromosome 13.
5. A cluster according to any of the preceding items, wherein the derived BS cells are xeno-free BS cells, such as a xeno-free hBS cells.
6. A cluster according to any of the preceding items, wherein the cluster is xeno-free.
7. A cluster according to any of the preceding items, containing from about 10 to about 5000 cells.
8. A cluster according to any of the preceding items, wherein said expression value of the up-regulated genes is about 750 or more such as, e.g., an expression value of about 1000 or more, about 1500 or more, about 2000 or more, about 2500 or more or about 3000 or more.
9. A cluster according to any of the preceding items, wherein said FC(CMLC) value of the up-regulated genes is about 20 or more such as, e.g., about 50 or more, about 100 or more, about 500 or more, about 750 or more, about 1000 or more.
10. A cluster according to items 2-9, wherein said ratio of FCCMLC/FCMC of the up-regulated genes is about 15 or more such as, e.g., about 20 or more, about 50 or more, about 100 or more, about 500 or more, about 750 or more, about 1000 or more.
11. A cluster according to any of the preceding items, wherein 2 or more such as, e.g., 4 or more, 6 or more, 8 or more, 10 or more, 12 or more or 16 or more of the up-regulated genes are genes associated with cardiac cells (described in Table II herein).
12. A cluster according to any of the preceding items, wherein 2 or more such as, e.g., 4 or more, 6 or more, 8 or more of the up-regulated genes are genes associated with endodermal cells (described in Table II herein).
13. A cluster according to any of the preceding items, wherein 2 or more such as, e.g., 4 or more, 6 or more, 8 or more of the up-regulated genes are genes associated with non cardiac or non endodermal cells, described in Table II herein.
14. A cluster according to any of the preceding items, wherein the up-regulated genes comprise 10 or more such as, e.g., 20 or more, 30 or more, 40 or more, 50 or more, 55 or more or all genes listed in Table II herein.
15. A cluster according to any of the preceding items, wherein the cluster comprising genes that are up-regulated and have,
i) expression values of 2000 or more,
ii) a FCCMLC value of 100 or more.
16. A cluster according to item 15, wherein the cluster comprising genes that are up-regulated and further have,
iii) a ratio between FCCMLC and FCMC of 100 or more.
17. A cluster according to items 15 and 16 derived from BS cells such as, e.g., hBS cells.
18. A cluster according to items 15-17, wherein the derived BS cells are trisomic hBS cells carrying an extra chromosome 13, such as SA002 carrying an extra chromosome 13.
19. A cluster according to items 15 to 18, containing from about 10 to about 2000 cells.
20. A cluster according to items 15-19, wherein said expression value of the up-regulated genes is about 2500 or more such as, e.g., an expression value of about 3000 or more, about 3500 or more, about 4000 or more, about 4500 or more, about 5000 or more, about 5500 or more, about 6000 or more, about 6500 or more, or about 7000 or more.
21. A cluster according to items 15-20, wherein said FC(CMLC) value of the up-regulated genes is about 200 or more such as, e.g., about 400 or more, about 600 or more, about 800 or more, about 1000 or more, about 2000 or more.
22. A cluster according to items 16-21, wherein said ratio of FCCMLC/FCMC of the up-regulated genes is about 150 or more such as, e.g., about 200 or more, about 250 or more, about 400 or more, or about 500 or more.
23. A cluster according to items 15-22, wherein 2 or more such as, e.g., 4 or more, or 6 or more, of the up-regulated genes are genes associated with cardiac cells (described in Table Ill herein).
24. A cluster according to items 15-23, wherein 2 or more such as, e.g., 4 or more, 6 or more, of the up-regulated genes are genes associated with endodermal cells (described in Table Ill herein).
25. A cluster according to items 15-24, wherein 5 or more of the up-regulated genes are genes associated with cardiac cells and 4 or more of the up-regulated genes are genes associated with endodermal cells (described in Table Ill herein).
26. A cluster according to items 15-25, wherein the up-regulated genes comprise 2 or more such as, e.g., 4 or more, or 6 or more, 8 or more or all genes listed in Table Ill herein.
27. A cluster according to any of items 1-26, wherein the cluster expresses one or more ion channels.
28. A cluster according to item 27 wherein the ion-channel is a K—, Na—, and/or Ca-ion channel.
29. A cluster according to item 27 or 28, wherein the ion channel is a K— or Na-voltage-gated channel, K— or Na-ligand-gated channel, a K-inwardly-rectifying channel and/or a Ca-voltage-dependent channel.
30. A cluster according to any of items 27-29, wherein the one or more ion channels are selected from the ion channels listed in
31. A cluster according to item 30 expressing at least 3 such as, e.g., at least 4, at least 5 or all of the ion channels listed in
32. A cluster according to any of the preceding items, wherein at least 4 of the genes associated with cardiac cell, at least 4 of the genes code for transcription factors and at least 4 of the genes code for ion channels listed in
33. A cluster according to item 32, where the genes are selected from the genes listed in listed in
34. A composition of cardiomyocyte-like clusters according to any of items 1-33, wherein at least 10%, such as e.g. at least 13%, at least 15%, or about 17% of the clusters contain nodal-like cells.
35. A composition of cardiomyocyte-like clusters according to any of items 1-34, wherein at least 30%, such as e.g. at least 35%, at least 40%, at least 45% or about 50% of the clusters contain atrial-like cells.
36. A composition of cardiomyocyte-like clusters according to any of items 1-35, wherein at least 20%, such as e.g. at least 23%, at least 26%, at least 30%, or about 33% of the clusters contain ventricle-like cells.
37. A composition of cardiomyocyte-like clusters according to any of items 1-36, containing a mixture of clusters containing nodal-like cells, clusters containing atrial-like cells and clusters containing ventricle-like cells.
38. A composition of cardiomyocyte-like clusters according to item 37, wherein the ratio between the number of clusters containing nodal-like cells and the number of clusters containing atrial-like cells is in a range of from about 1:100 to about 50:100 such as from about 10:100 to about 40:100, from about 20:100 to about 40:100, from about 30:100 to about 40:100 or about 33:100-34:100.
39. A composition of cardiomyocyte-like clusters according to item 37, wherein the ratio between the number of clusters containing nodal-like cells and the number of clusters containing ventricle-like cells is in a range of from about 1:100 to about 80:100 such as from about 10:100 to about 70:100, from about 30:100 to about 70:100, from about 45:100 to about 55:100 or about 50:100.
40. A composition of cardiomyocyte-like clusters according to item 37, wherein the ratio between the number of clusters containing ventricle-like cells and the number of clusters containing atrial-like cells is in a range of from about 1:100 to about 90:100 such as from about 40:100 to about 80:100, from about 50:100 to about 75:100 or about 66:100.
41. A composition of cardiomyocyte-like clusters according to item 37, wherein the ratio between the clusters containing nodal-like cells, the clusters containing atrial-like cells and the clusters containing ventricle-like cells is 17:50:33.
42. A composition comprising one or more clusters as defined in any of items 1-33 and a carrier.
43. A composition according to item 42, wherein the carrier is an aqueous medium.
44. A composition according to item 42 or 43 in liquid or frozen form.
45. A composition according to any of items 42-44, wherein the aqueous medium contains one or more additives.
46. A composition according to item 45, wherein the additive is one or more cryoprotectants, one or more stabilizers and/or one or more viscosity-adjusting agents.
47. A composition according to any of items 42-46, wherein the one or more cryoprotectants is selected from the group consisting of ethylene glycol, propylene glycol, dimethylsulfoxide, glycerol, propanediol, and methyl pentanediol, and or mixtures thereof.
48. A composition according to any of items 42-47, wherein the one or more additive is a sugar or sugar alcohol including sucrose, trehalose, maltose, lactose
49. A composition according to any of items 42-48, wherein the cryoprotectant is trehalose.
50. A composition according to item 49, wherein the concentration of trehalose is from about 0.02 M to about 1 M, such as, e.g., from about 0.05 M to about 0.9 M, from about 0.1 M to about 0.8 M, from about 0.15 M to about 0.7 M, from about 0.2 M to about 0.65 M, from about 0.25 M to about 0.6 M.
51. A composition according to any of items 42-50, wherein the cryoprotectant is sucrose.
52. A composition according to item 51, wherein the concentration of sucrose is from about 0.02 M to about 1 M, such as, e.g., from about 0.05 M to about 0.9 M, from about 0.1 M to about 0.8 M, from about 0.15 M to about 0.7 M, from about 0.2 M to about 0.65 M, from about 0.25 M to about 0.6 M.
53. A composition according to any of items 34-52, wherein the cryoprotectant is DMSO.
54. A composition according to any of items 34-53, wherein the concentration of DMSO is at least 2.5% v/v, such as e.g. from about 2.5% to about 40% v/v, from about 5% to about 35% v/v, from about 7% to about 30% v/v, from about 7% to about 25% v/v, from about 7% to about 20% v/v, from about 15% to about 25% v/v, or from about 5% to about 15% v/v.
55. A composition according to any of items 34-53, wherein the cryoprotectant is ethylene glycol.
56. A composition according to item 55, wherein the concentration of ethylene glycol is at least 2.5% v/v, such as e.g. from about 2.5% to about 30% v/v, from about 5% to about 25% v/v, from about 5% to about 20% v/v, from about 10% to about 20% v/v, from about 7% to about 10% v/v, or from about 2.5% to about 5% v/v.
57. A composition according to any of items 34-56, wherein the viscosity-adjusting agent is selected from the group consisting of Ficoll, Percoll, hyaluronic acid, albumin, polyvinyl pyrrolidone, alginic acid, gelatin and glycerol.
58. A composition according to any of items 34-57, wherein said viscosity-adjusting agent is Ficoll.
59. A composition according to item 58, wherein the concentration of Ficoll is at the most about 150 mg/ml, such as, e.g., at the most about 100 mg/ml, at the most about 50 mg/ml, at the most about 25 mg/ml, at the most about 15 mg/ml or at the most about 10 mg/ml.
60. A composition according to any of items 34-59, wherein the one or more clusters retain at least 95% of its characteristics as defined in any of items 1-33 after storage of the composition at a temperature of at least −80° C. for at least 2 years.
61. A composition according to any of items 34-60, wherein about 50% or more such as, e.g., about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more or about 95% or more of the cells are viable after storage of the composition at a temperature of at least −80° C. for at least 2 years.
62. A kit for use in testing of cardiotoxicity of a specific substance, the kit comprising
i) one or more clusters as defined in any of items 1-33 or a composition as defined in any of items 34-61,
ii) specific instructions for use of the cluster or composition, whichever is relevant.
63. A kit according to item 62 further comprising
iii) a medium into which the specific substance is dispersed before use of the kit.
64. A kit according to any of items 62 or 63 comprising a composition as defined in any of items 34-61.
65. A kit for use in in vitro testing during drug discovery of a specific substance, the kit comprising
i) one or more clusters as defined in any of items 1-33 or a composition as defined in any of items 34-61,
ii) specific instructions for use of the cluster or composition, whichever is relevant.
66. A kit according to item 65 further comprising
iii) a medium into which the specific substance is dispersed before use of the kit.
67. A kit according to any of items 65 or 66 comprising a composition as defined in any of items 34-61.
68. A kit for regenerative medicine comprising
i) a composition as defined in any of items 34-61 and/or one or more cardiomyocyte-like cell clusters as defined in any of items 1-33,
ii) tools for administration of the composition or the cells to a patient such as, e.g., the cells in an administrative form, such as in a ready-to-use syringe.
69. A kit according to item 68 comprising a composition as defined in any of items 34-61.
70. A method for obtaining one or more clusters according to items 1-33, comprising 3D formation of differentiating hBS cells in a medium supplemented with one or more members from the transforming growth factor beta superfamily and/or one or more members from the fibroblast growth factor family.
71. A method for obtaining the clusters according to items 1-33 comprising the steps of,
72. A method for obtaining the clusters according to items 1-33 comprising the steps of,
73. A method according to any of items 71 and 72, wherein the suspension of primarily undifferentiated hBS cells in step 1 are obtained by enzymatic treatment.
74. A method according to any of items 71-73, wherein the dissociation in step 3) is performed manually.
75. A method according to any of items 72-74, wherein the centrifugation is performed at 100-800×g for 2-20 minutes, such as e.g. at 200-600×g for 5-10 minutes, or 300-500×g for 5-10 minutes, or at 400×g for 5 minutes.
76. A method according to any of items 72-75, wherein the sedimentation is performed for about 1-36 hours, such as e.g. for about 2-24 hours or for about 3-12 hours.
77. A method according to any of items 72-76, wherein the cells obtained after sedimentation and/or centrifugation are incubated for 1-10 days in step 5), such as e.g. 1-7 days or 1-5 days, for the formation of 3D structures.
78. A method according to any of items 72-77, wherein the cells obtained after sedimentation and/or centrifugation are incubated 3 days in step 5), for the formation of 3D structures.
79. A method according to any of items 71-78, wherein the development in step 5) of item 71 and/or step 7) of item 72 are for 1-30 days, such as e.g. 1-15 days, 1-10 days, or 1-5 days, until the formation of cardiomyocyte-like cell clusters.
80. A method according to any of items 72-79, wherein the medium used in step 5) and/or 7) of item 72 comprises 15-25% of FBS, such as e.g. 20% FBS.
81. A method according to any of items 71-80, wherein the medium used in step 2) of item 71 and/or step 5 and/or 7) of item 72 comprises a member from the transforming growth factor beta superfamily and/or a member from the fibroblast growth factor family.
82. A method according to item 81, wherein the member from the transforming growth factor beta superfamily is Activin A and the member from the fibroblast growth factor family is bFGF.
83. A method according to item 82, wherein the concentration of Activin A supplemented to the culture medium is about 5-40 ng/ml, such as e.g. 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, or 12 ng/ml.
84. A method according to item 82, wherein the concentration of bFGF supplemented to the culture medium is about 5-40 ng/ml, such as e.g. 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml or 14 ng/ml.
85. A method according to item any of items 71-84, wherein the medium used in step 2) of item 71 and/or step 5 and/or 7) of item 72 comprises Activin A, bFGF and/or FBS.
86. A method according to any of items 72-85, wherein the medium used in step 5 and/or 7) of item 72 comprises a member of GSK-3 inhibitors and/or p38 MAP kinase inhibitors.
87. A method according to item 86, wherein the member of the GSK-3 inhibitor family is SB 216763 and the member of the p38 MAP-kinase inhibitor family is SKF-860002.
88. A method according to any of item 86 and 87, wherein the concentration of the GSK-3 inhibitor supplemented to the culture medium is from about 1-25 μM, such as e.g. from about 2-10 μM or about 5 μM.
89. A method according to any of item 86-88, wherein the concentration of the p38 MAP-kinase inhibitor supplemented to the culture medium is from about 1-25 μM, such as e.g. from about 2-10 μM, or about 5 μM.
90. A method according to any of items 71-89, wherein medium used in step 5) of item 71 and/or step 7) of item 72 comprises between 100-2000 U/ml LIF, such as e.g. between 500-1500 U/ml, or 1000 U/ml.
91. A method according to item 90, wherein the medium comprising LIF is replaced after 2-8 days of incubation, such as after e.g. 4-5 days, with a medium without LIF.
92. A method according to any of items 71-91, wherein the culture disc(s) in step 5) of item 71 and/or step 7) of item 72 has a gelatine coated surface.
| Number | Date | Country | Kind |
|---|---|---|---|
| PA 2007 01345 | Sep 2007 | DK | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2008/007835 | 9/18/2008 | WO | 00 | 6/10/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60960160 | Sep 2007 | US | |
| 60996768 | Dec 2007 | US | |
| 61129177 | Jun 2008 | US |
