[0001] A METHOD OF PREPARING ISOLATED CELLS AND USES
[0002]This invention relates to cells differentiated from induced pluripotent stem cells (iPSC) that have been isolated from a source, a method for differentiation and uses of the differentiated cells thereof. More particularly it relates to a method of differentiating human induced pluripotent stem cells (iPSC's) to form cardiac cells (including progenitor cells that retain haematopoietic potential) and to the use of such cells in medicine or screening drugs.
[0004] Mortality rates are high for ischaemic heart disease (1 ). As the adult heart is unable to rapidly regenerate, a large number of cardiomyocytes are lost immediately after infarction. Thus, patients are left with an area of irreversibly damaged myocardium. Stem cells and their products have the potential to provide or stimulate the formation of a new blood supply and new viable cardiomyocytes within the damaged heart, thereby reducing the hemodynamic stress and left ventricular remodelling that occurs post trauma. Reduction of such stress and remodelling can reduce the risk of heart failure. Cellular therapies using human post-natal blood and bone marrow have been tested in clinical trials for cardiac regeneration and, although randomised clinical trials show some evidence of improvement in left ventricular ejection fraction over the short term, there is still no conclusive evidence of cardiomyocyte replacement with these cells (2-5). Recently identified cardiac stem cells (CSCs) offer a promising cell population for autologous cell therapy or for developing drug therapeutics for endogenous repair (6, 7). However, human cardiac biopsy samples are required and this lack of readily available tissue limits studies on autologous CSCs in patients susceptible to myocardial infarction. Human embryonic stem (ES) cells remain one potential source of cells for regenerating infarcted tissue with functioning cardiomyocytes and their ability to contribute to cardiac repair following myocardial infarction has been described in detail (8-12). However their derivation from live embryos is
considered ethically contentious and problems related to donor-recipient mismatch should not be underestimated (13).
[0005] In ground breaking studies, Yamanaka and colleagues have identified the genetic factors that can reprogram somatic cells to an embryonic-like state (14). These cells, termed induced pluripotent stem (iPS) cells, have been generated in vitro from adult mouse (14) and subsequently human tissue sources (15-17). Being derived from host somatic cells, they also represent a source of autologous cells for therapy and research. Other technical advances, such as the use of non-integrating viral vectors (18, 19) or small molecules (20-22) have been reported, but the therapeutic potential of such human iPS lines for cardiac repair has yet to be confirmed.
[0006]Currently, only limited in vivo studies describe the potential of human iPS cells for cardiac repair (23), and report the effects of directly injecting undifferentiated iPS cells into infarcted hearts of immuno-suppressed and immuno-competent mice. Treatment of immuno-competent hearts resulted in successful cell engraftment and an improvement in cardiac function. However, undifferentiated iPS cells administered to immuno-suppressed hearts formed tumours over 2-4 weeks.
[0007] Differentiation of iPS cells to haematopoietic progenitors and subsequently red blood cells, generally relies on embryoid body formation, and media containing animal products (i.e. serum) which are not produced to GMP standards. This makes production of haematopoietic progenitors and red blood cells for diagnostics and transfusion medicine difficult, since embryoid body formation is not amenable to scale-up for manufacture, and use of media that are not GMP compliant means cells derived may not be approved for use in the clinic. Notably these approaches also result in an inefficient and ill-defined differentiation process that then requires complex steps to purify resulting haematopoietic progenitors. The present invention seeks to address some of the issues.
[0008]According to one aspect of the present invention there is provided a method for differentiating induced pluripotent stem cells comprising the steps of
a) subjecting the induced pluripotent stem cells to a first media comprising a cytokine or growth factor for a first period,
[0009] b) replacing the media from step a) with a second media comprising a combination of cytokines or growth factors for a second period, c) changing the media after step (b) and adding a third media with no added cytokines or growth factors and incubating for a third period to provide differentiated cells.
[0010]Alternatively, if haematopoietic cells are to be derived, step (c) may be replaced by step (d) where the media after step (b) is changed and a fourth media added which has added cytokines and growth and incubated for a fourth period.
[0011]Steps (a) to (c) may result in cardiac progenitor cells, cardiac myocytes, smooth muscle cells, fibroblasts, endothelial cells, haematopoetic cells and cardiac progenitor cells that retain haematopoietic potential.
[0012]Replacing step (c) with step (d) gives rise to haematopoietic cells. Preferably, the cytokine in the first media in step (a) is activin A and may be provided in a concentration of less than 100ng/ml, or between 40ng/ml and 60ng/ml for example 50ng/ml. However, it will be understood by a person skilled in the art that any suitable concentration may be used. Preferably the second media comprises Activin A , bFGF and BMP4.
[0013]All the media may comprise serum free media or any other suitable media for the growth or culture of stem cells. All or any of the media may be GMP (Good Manufacturing Practice) compliant. An example of such a media is StemPro34 (Invitrogen) which is GMP compliant. The first, second, third or fourth media may be supplemented by the addition of one or a combination of Penicillin, Streptomycin, L-glutamine, monothioglycerol, ascorbic acid, bovine serum albumin and fetal calf serum
Glutamine, Penicillin and Streptomycin are standard reagents in the maintenance, culture and growth of stem cells. Monothiologlycerol is used for stem cell maintenance, whilst ascorbic acid can help to promote differentiation towards particular cells types.
[0014]The first period may be less than 24 hours and preferably between 1 and 12 hours or 4 hours. The second period may be between 4 hours to 6 days, in particular between 24 and 48 hours, or 96 hours. The third and fourth periods of incubation and culturing may be between 2 days and 21 days, particularly between 4 days and 14 days. It will be understood by a person skilled in the art that any period suitable for cell culture may be used.
[0015]In steps (b) the combination of cytokines or growth factors may be selected primarily from activin A, bFGF (basic factor growth factor), BMP4 (bone morphogenic protein), VEGF (vascular endothelial growth factor) and DKK (dickaopf), as well as small molecules that may act similarly to cytokines and growth factors to promote activation of activin A, bFGF, BMP4 or modulate the associated signalling pathways. Other pathways that maybe modulated to further improve yield of cardiovascular cells may include but is not confined to cytokines, growth factors and small molecules that regulate the VEGF signalling, Wnt signalling, Notch signalling and Hedgehog signalling pathways For example, a combination of the cytokines activin A, bFGF and BMP4 may be used. BMP4 and dFGF are growth factors typically used to promote mesodermal and cardiovascular differentiation, at many various different concentrations and combinations. For example, Laflamme et al (10) use only BMP4 in subsequent differentiation after high dose of activin A. In the present method, lower doses of activin A are used with both BMP4 and bFGF. Advantageously, this particular combination on iPSC monolayers gives rise to the sheets of contractile tissue. The culture period may be around 14 days.
[0016]Preferably, the concentration of each cytokine or growth factor may be between 1 and 100 ng/ml, for example 10ng/ml each.
In step (d), for making haematopoietic cells, primarily VEGF (vascular endothelial growth factor) but also other growth factors, cytokines or small molecules that may induce haematopoiesis from endothelial cells, such as those that induce RUNX1 , may be used either alone or in combination. Other pathways that maybe modulated to further improve yield of haematopoietic cells include but are not restricted to those affecting Wnt signalling, Notch signalling and Hedgehog signalling pathways.
[0017]The fourth media may be added from day 4- day 8 to initiate a haematopoietic programme, for the induction of haematopoietic cells from haemogenic endothelium within the cardiac cultures.
[0018]The inventors have demonstrated for the first time that cardiac progenitors formed in the present method retain a haematopoietic potential, such that upon application of appropriate conditions, they form erythroid and myeloid cell types, via a 'haemogenic' endothelium. As such this method is applicable to derive both cardiovascular and haematopoietic lineages.
[0019]This directed approach to derive cardiac and haematopoietic progenitors, relies on differentiation of iPS cells as a monolayer, results in efficient cardiac differentiation which retains the potential to form haematopoietic progenitors. Further, the method is amenable to scale up and manufacture. Resulting haematopoietic progenitors that arise can be further expanded without complex purification, and so is cost effective and suitable for scale-up and automation. Resulting haematopoietic cells could also be manufactured to GMP standards. Notably, most processes described to-date are for differentiation of human embryonic cells and do not cover haematopoietic differentiation from human induced pluripotent stem cells. Since the method involves deriving haematopoietic cells from cardiac cultures via a haemogenic endothelium, in a process that mirrors 'definitive' or adult haematopoiesis, it is more likely to yield haematopoietic progeny that will be useful in diagnostics, testing and transfusion and transplantation medicine
Pluripotent stem cells may be induced from human dermal fibroblasts or any other source of human tissue, embryonic or adult, from any part of the body These cells may be isolated cells from the source and engineered appropriately to be then further differentiated in vitro as appropriate. Induced Pluripotent Stem cells (or iPS cells) may be a pluripotent stem cell (one that can contribute to all tissues of the body) that has been engineered or induced, from non-pluripotent stem cells, that being from adult or embryonic tissue which is developmental^ 'restricted' and no longer considered pluripotent, i.e. tissue specific stem cells or differentiated somatic cells. Engineering of induced pluripotent stem cells is well known in the art and can take the form of transgene expression, or use of small molecules, including pharmaceuticals, genetic material, or proteins, to manipulate as necessary.
[0020]In one embodiment, the induced pluripotent stem cells may be differentiated as a monolayer for a complete time course over 14 days in a supplemented media ( such as StemPro34, Invitrgen) comprising 50 ng/ml activin A for 4h; and utilising the same supplemented media with reduced levels of activin A in step (b) compared to step (a) at around 5ng/ml activin A as well as bFGF 5ng/ml, BMP4 10 ng/ml for upto 96h with at least one full media change with fresh cytokines; and utilising a serum free media such as StemPro34 where no cytokines or growth factors have been added, from 96h to 14-21 days until the cells start to beat. It will be appreciated by a person skilled in the art that the invention is not limited by these parameters. These methods may be carried out in vitro. Preferably, the method may be conducted in the absence of serum.
[0021]The method can be used to produce differentiated cardiac cells which may include cardiac progenitor cells and cardiac proginator cells that retain a haematopoietic potential, cardiac myocytes, fibroblasts, smooth muscle cells, endothelial cells, and haematopoietic progenitors. In particular the differentiated cardiac cells may include beating cardiac myocytes. The differentiated cardiac cells may be in the form of sheets which may have several layers of cells. Cardiac cells may include cardiac progenitors derived
at earlier time points, that retain a haematopoietic potential, or specific cardiac cell types i.e. cardiomyocytes, smooth muscle cells, endothelial cells, fibroblasts, and haematopoietic progenitors from a given period such as day 14-21 of culture or incubation.
[0022]In a further aspect, there is provided a heterogeneous population of cells derived from induced pluripotent stem cells (iPSC) comprising one or more of cardiac progenitor cells, cardiac myocytes, smooth muscle cells, fibroblasts, endothelial cells, and haematopoietic cells.
[0023]The cardiac progenitor cells and/or cardiac monocytes stain for platelet derived growth factor a receptor (PDGFaR) on day 6 and/or stain for a actinin, troponin I, and / or troponin C on day14. The smooth muscle cells stain for a smooth muscle actin. The endothelial cells may stain for CD34 and/ or CD31 . The haematopoietic progenitor stains positively for CD45.
[0024]The cardiac myocytes and / or cardiac progenitor cells may comprise at least 10%, through 20%, 30%, 40%, 50%, 60%, 70%, 80% to as 90+% of the total heterogeneous cell population. Preferably the heterogeneous population of cells comprise at least 30% cardiac cells and / or cardiac progenitor cells of the total cell population. For example, the cell population may comprise 38% cardiac progenitor cells on day 6, giving rise to 39% cardiomyocytes, on day 14.
[0025]With the addition of VEGF on day 4 to day 8, haematopoietic cells are induced and emerging from subsequent culture in haematopoietic conditions, which demonstrates the existence of haemogenic endothelium in this method.
[0026]The population may also preferably comprise other differentiated cell types in the following ranges: 6% (2- 20%) smooth muscle cells and 1 .66% vascular cells (1 -20).
The heterogeneous cell population may be further purified or differentiated such that they are wholly cardiac progenitors (retaining haematopoietic potential) or cardiomyocytes cells. It is possible to derive from this heterogeneous population of cells, cardiovascular progenitor cells or progeny of these i.e. cardiomyocytes, endothelial cells, smooth muscle, fibroblasts and haematopoietic cells.
[0027]According to another aspect of the present invention there is provided a kit or composition comprising either:
[0028]a. A heterogeneous population of cells derived from induced pluripotent stem cells (iPSC); or
[0029]b. Differentiated cardiac myocytes or cardiac progenitor cells, or cardiac proginator cells with haematopoietic potential; or
[0030]c. One or more sheets of cells comprising beating cardiac myocytes; or d. Haematopoietic progenitors in suspension and progeny including red blood cells and myeloid cells.
[0031]The cells or sheets may be provided on a pharmaceutically acceptable carrier or medium. Examples include, but are not limited to, bandages, scaffolds, stents, and gel. The cells may be provided on a surface or embedded in the carrier, for example.
[0032]According to a further aspect of the present invention there is provided a heterogeneous population of cells derived from induced pluripotent stem cells (iPSC) or differentiated cardiac cells or cardiac progenitor, or cardiac progenitors with haematopoietic potential for use in medicine. Such uses include but are not limited to cardiac disease or wound healing, transfusion medicine and diagnostics. More particularly the cardiac disease to be treated may be myocardial infarction, congenital heart conditions, heart valve replacement, refractory angina/ischemia and heart failure. The cells or sheets according to the present invention may be used in the treatment or prevention of other conditions as appropriate. The haematopoietic cells obtained by the present method can be used in therapies such as, red blood cell and platelet
transfusion, neutrophil and dendritic cell therapies, other transplantation regimes including macrophage, T and B cell transplantation and novel diagnostics such as antibody testing panels. According to a another aspect of the present invention there is provided a method of treating cardiac disease comprising administering a heterogeneous population of cells derived from induced pluripotent stem cells (iPSC) or differentiated cardiac cells comprising cardiac progenitor cells and/or cardiomyocytes in a therapeutic amount to a patient in need thereof. A therapeutically effective amount may comprise at least 1 x106 cells/kg of body weight.
[0033]Another aspect is to provide differentiated iPSC cells and methods for their production which may be used for research, pharmaceutical testing and therapies without the risks associated with the use of undifferentiated iPSCs. One of the advantages of this invention is that it can be done with such high efficiency in One dish', where cells are maintained from start to finish, in the same dish without the need for complex purification or isolation strategies, and thus permits scale up and manufacture. The fact that sheets of cardiac cells with beating cardiomyocytes can be produced is particularly useful for pharmaceutical testing of drugs. Further, earlier progenitor cells, especially those obtained at day 6 and those that retain haematopoietic potential may be useful for regenerative therapy such as myocardial infarction, congenital heart conditions, heart valve replacement, refractory angina/ischemia and heart failure as well as haematopoietic progenitors being useful for transfusion and diagnostics such as red blood cell and platelet transfusion, neutrophil and dendritic cell therapies, other transplantation regimes and novel diagnostics such as antibody testing panels. One dish' is taken to mean where cardiac cells (and progenitors with haematopoietic potential) are maintained and differentiated, from a starting population of iPS cells to cardiovascular and haematopoietic cells, over a given period, such as 14-21 days, in one dish (or as a single monolayer
and/or sheet of cells) without the need for complex purification or isolation strategies.
[0034]To date, most iPS research has focused on the derivation of cardiac progenitors from mouse iPS cells (38, 39) and generally this has been to assess differentiation capacity in vitro. Whilst several studies have demonstrated human iPS differentiation towards the cardiac lineage (15, 31 ), these protocols have not been extended to demonstrate the existence of haematopoietic cells arising from haemogenic endothelium or where long- term engraftment of human iPS cells has been reported in murine models of myocardial infarction (23), these studies involved the transplantation of undifferentiated human iPS cells into immuno-suppressed mice and resulted in formation of tumours. The present highly efficient differentiation protocol for the production of cardiovascular cells from human iPS cells as monolayers provides alternatives to what is known in the art, and is extremely novel in demonstrating that cardiac progenitors retain haematopoietic potential, with haematopoietic cells arising from a haemogenic endothelium. When cardiac differentiated cells were transplanted at day 6 of differentiation in a rat model of myocardial infarction, these cells were shown to be capable of engraftment and to persist long term, with further differentiation into cardiovascular cell types in vivo. Although the protective effects from injection of the iPS-derived cardiac cells were generally modest after myocardial infarction, there was a non-significant trend in decline of ejection fraction at 10 weeks (p<0.2), when compared to the control infarcts. Previously, mixed populations of differentiating human embryonic stem cell cultures have been used (10, 1 1 ) as it is thought that contaminating cells such as fibroblasts may actually improve transplant efficiency by supporting engraftment of the cardiac progenitors (12). Of note, is that even with a mixed differentiated population, no teratoma formation was observed, and there
were no tumour related mortalities. This helped to alleviate safety concerns associated with such a procedure. Purified populations of human embryonic stem cell derived cardiomyocytes have been used with success to promote cardiac repair (10), but this is separate to work described herein for iPS cells.
[0035]Novel methods for delivery in appropriate animal models, are described in Bell et al. (41 ) using primate ES cells, with their cardiac differentiated progeny introduced via composite sheets prior to implantation. The inventors have shown, for the first time, that human iPS derived cardiac progenitors can be derived at high efficiency using directed differentiation as monolayers.
[0036] Cardiac progenitors retain an ability to form haematopoietic cells, via a haemogenic endothelium. When induced with VEGF on day4-8 and subsequently expanded in haematopoietic culture, myeloid and erythroid cells arise.
[0037] When injected as progenitors into the infarcted rat heart, they were able to engraft, differentiate into cardiomyocytes and smooth muscle, and persist long-term.
[0038]Furthermore, compared to control infarcted hearts, the iPS-derived cells ameliorated the reduction in ejection fraction at an extended time frame of 10 weeks. Although this effect at 10 week was small and non-significant, this does suggest a contribution from functional cardiomyocytes, as these had been shown to be contractile from day 12, with, potentially, an additional contribution from paracrine mediated protection from re-modelling over this time.
[0039] That they were produced efficiently as monolayers, in a serum- and feeder- free, directed approach means we have gone some way to addressing a major limitation to the field, that is GMP compliant manufacture and scale up where the availability of human clinical grade tissue for heart repair will not be in short-supply. Furthermore, this protocol opens up the possibility of having a ready supply of red blood cells for transfusions and other therapeutic or research uses.
The invention will be described by way of illustration only with reference to the following Examples and the accompanying drawings, in which:
[0040]Figures 1 a) to c) illustrate directed differentiation of iPSC's using a protocol according to one aspect of the invention; a flow chart of the protocol is outlined in a); top, with bright field images (x4) that clearly reflect the drastic loss of iPS morphology, as cells differentiate towards the cardiovascular lineage, shown below in the photographs in a); On days 4-8, populations were assessed after harvesting, by FACS analysis after staining for the expression of Tie2, c-kit, and PDGFRa as shown in b) (where n=3); with subsequent loss of embryonic stem cells markers TRA-1 -81 and SSEA4 shown in c). Figures 2 a) to j) illustrate differentiated cells by phenotypic assessment;
[0041]After 14 days of differentiation, iPS cells were stained for a range of cardiovascular markers that include a) a-actinin and Troponin C (x10) with b) showing a magnified (x60) image and inset showing striations that indicate sarcomeric staining for α-actinin c) shows CD34 with CD31 (x20) and d) showing a magnified (x40) image of these double positive networks; e) shows Troponin C staining with CD31 staining demonstrating association of endothelial networks with cardiomyocytes (x20); f) shows smooth muscle actin (green) with CD31 staining (x20) with a magnified image demonstrating SMA specific cytoskeletal staining in inset. Quantification of these biomarkers after harvest of cells from culture on day 14, and analysis by FACS as indicated (n=4-8 as indicated ) are shown in the graphs with g) showing Toponin I, h) alpha-actininin, i) smooth muscle actin (SMA) and j) CD31 staining. Figures 3A) to F) illustrates the effect of administering iPSC's on heart function following myocardial infarction; RNu rats were monitored from day 2 to week 10 following myocardial infarction using in vivo MRI. Fig3A is representative MR images, taken at 10 weeks post Ml, of sham-operated hearts (top), infarcted hearts (middle) and iPS-treated infarcted hearts
(bottom) at diastole (left) and systole (right); (scale bar = 10 mm; arrows indicate infarcted region); (B) Mean end diastolic; C) end systolic; D) ejection fractions; E) relative infarct; F) mean mass from sham (clear), infarcted (horizontal lines) and iPS-treated (diagonal lines) -hearts show that iPS- derived cell administration attenuated the decrease in ejection fraction at 10 weeks compared to infarct controls ( p < 0.2 10 weeks). This was also true for end systolic volume (ESV) (p=0.2) and average LV mass (p<0.2). Significance between infarct and sham groups is also shown where * denotes p<0.05. ; and Figures 4a) to c) illustrates cell engraftment 10 week post myocardial infarction. After 10 weeks post infarction, rat heart tissues from iPS injected infarcts were collected, fixed and sectioned. They demonstrate: a) direct GFP fluorescence (green) and immuno-fluorescence staining for human Troponin I (red), or immuno-fluorescent staining for GFP with b) aActinin and c) SMA positive cells (all at x40) as indicated. In each case the left hand panel shows counter staining with DAPI, middle left panel shows GFP expression, middle right shows the relevant cardiovascular stain, and the right panel shows a merged imaged to demonstrate co-localisation. Upper panels in a), b) and c) are representative sections from infarct control groups, whilst the lower panels are from those infracted hearts injected with GFP positive iPS cells differentiated towards the cardiac lineage.
[0042]Fig 5a) to g) illustrates the emergence of haematopoietic cells from cardiac cultures, when stimulated on day4-8 with VEGF, and subjected to standard haematopoietic expansion conditions. In a) we show staining for CD31 (red) and CD45 (green) in cardiac culture after exposure to VEGF (10ng/ml) from day 4-8. Cells co-stain for both markers, which can be used to identify the haemogenic endothelium. This is further confirmed by FACS analysis (b-c) where a double positive population (marked as p2) is observed in controls (b) and which can be induced after exposure to VEGF (c). CD31 positive cells were isolated by MACS separation on CD31 , haematopoietic cells can form CFU colonies in methocult culture where CFU-G are shown in d) CFU-M in e) and where monocytes are stained red for CD14 and green for CD45 in f). In g) erythroid cells (identified with arrows) can also be seen to form in cardiac
cultures when supplemented with haematopoietic factors SCF, IGF, EPO and Dexamethasone.
[0044]EXAMPLE 1 - Derivation and culture of human iPS cells:
[0045] iPS cells were generated essentially as described by Takahashi et al.(15) and Carpenter et al (24). Briefly, human dermal fibroblasts (Lonza, Tewkesbury, UK), were reprogrammed using retrovirus expressing Oct4, Sox2 and Klf4. Retrovirus was generated from pMX based plasmids (Addgene, Cambridge, USA) and PLATGP packaging cells (Cell Biolabs. , San Diego, USA). Transduced HDNF were co-cultured with Mitomycin C treated mouse embryonic fibroblasts in DMEM/F12, 20% (v/v) Knock Out Serum Replacement, 10 ng/ml bFGF, 2mM sodium pyruvate, 1x Non-Essential Amino Acids (Invitrogen Ltd. , Paisley, UK) and 1 μΜ beta-mercaptoethanol (Sigma-Aldrich Ltd. , St. Louis, Miss. , USA). Cultures were maintained in 5% CO2 and 5% oxygen with half media changes every 2 days, for up to 34 days. iPS colonies were picked and expanded prior to adaptation to feeder independent culture as described by Ludwig et al. (25), whilst cultured routinely in 5% oxygen. iPS colonies were harvested by day 5-6, when compacted, with 2 hours pre-incubation in Y27632 ROCK inhibitor (10 microM final) (Sigma Aldrich Ltd. , St. Louis, Miss, USA) as previously described (26). iPS cells were washed in DMEM/F12 basal media (Invitrogen Ltd. , Paisley, Scotland) and incubated in 1 x dispase (Invitrogen Ltd) for 20 min, after which cells were harvested, dispersed mechanically into clumps of 10-20 cells, and dispersed at ratios of 1 : 12 onto Matrigel (ES qualified Matrigel from BD Biosciences, Oxford, UK) coated plates with 2 ml fresh mTeSR (Stem Cell Technologies) supplemented with Y27632. Further feeds involved half media changes every 48 hours, without the need to further supplement with ROCK inhibitor. iPS cells used in this study, referred to as the C18 line, have been fully characterized for ES-like morphology, transgene silencing, ES specific gene expression and pluripotency in vitro with the differentiation towards neurons, hepatocytes and muscle, and in vivo as teratomas (24).
EXAMPLE 2 - Optimised Protocol for Cardiac Differentiation of iPS cells:
[0046]The inventors assessed various protocols previously described (10, 27) (data not shown) and compared them with their own differentiation strategy described. In contrast, the inventor's protocol (Fig 1 a) yielded sheets of contractile tissue, and this is described herein. iPS cells were differentiated as a monolayer in: Stage 1 : Stem Pro-34 medium (Invitrogen Ltd.) with 2 mM L- glutamine, 1 % (v/v) PenStrep (PAA Ltd. , Swindon, UK), 400 μΜ monothioglycerol (Sigma Aldrich Ltd.) and 50 pg/ml ascorbic acid (Sigma Aldrich Ltd.), with 50 ng/ml activin A for 4h; Stage 2: 10 ng/ml BMP4 (R & D systems, Abingdon, Oxfordshire, UK), 5 ng/ml bFGF, and 5 ng/ml Activin A (Invitrogen Ltd.) for 44 h; with a full media change, with fresh cytokines, at day 2-4; Stage 3: Stem Pro 34 basal medium alone with no added cytokines at day 4-14. Cell masses started beating after 10 days. Activin A, BMP4 and dFGF are all growth factors typically used to promote mesodermal and cardiovascular differentiation, but many various different concentrations and combinations have been described without the One dish' efficiency that was observed by the inventors. By "one dish" it is meant where cardiac cells are maintained and differentiated, from a starting population of iPS cells to cardiovascular cells, over a given period, such as 14-21 days, in one dish (or as a single monolayer of cells) without the need for complex purification or isolation strategies. In the prior art methods, activin A is used at much higher doses for a longer period (10). Also, in the prior art methods, BMP4 is used only in subsequent differentiation after high dose of Activin A. Surprisingly, the inventors have shown that a lower dose of Activin A with both BMP4 and bFGF provides the desired result not previously demonstrated. The present method allows the formation of the sheets of contractile tissue. The contractile sheets can be seen after 14 days. EXAMPLE 3 - Characterizing cardiovascular progenitor cells:
[0047] To assess whether the iPS cells contributed to the cardiac lineage and were capable of adult cardiogenesis, the C18 iPS cell line were cultured in the optimized protocol in Example 2, to first identify the mesoderm and cardiac progenitor phenotype. As described by Yang et al., for hES cells (27), cKit"
KDR (Flk 1 ) cardiac progenitors can be isolated and developed into cardiomyocytes. Additionally, PDGFalphaR is expressed during cardiovascular development (28), and thus may be considered an additional marker for the cardiac progenitor. Initially cells were collected after dispersal in accutase (PAA Ltd) for approximately 10 min during day 4-8 differentiation, pelleted at 300 g for 10 min, re-suspended in 90 μΙ PBS with 0.5% (w/v) BSA and mixed with 10 μΙ FcR blocking reagent (Miltenyi Biotec, Bergisch- Gladbach, Germany) for 15 min on ice. Next, live cells were stained for KDR- PE (R&D Systems), ckit-APC (BD Biosciences), Tie2-APC (R&D Systems), and PDGFRD-PE (BD Biosciences), by applying antibodies at 1 :5-1 : 10 dilution for 1 h at 4°C. After washing 3 times with PBS and pelleting at 1200 rpm for 10 min, the cells were analysed using an LSRII flow cytometer (BD Biosciences). EXAMPLE 4- Demonstrating cardiac and endothelial potential of human iPS cells:
[0048] Day 4-21 iPS cell differentiation cultures were fixed in 4% (w/v) formaldehyde/PBS for 15 min on ice, washed 3 times in PBS and incubated with PBS-BT (0.5% (w/v) BSA and 0.1 % (v/v) Triton X-100) at 4°C overnight prior to staining with relevant antibodies. Fixed cells were stained for the cardiac phenotype using troponin I (clone EP1 106Y, Abeam Ltd., Cambridge, UK), troponin C (clone H-1 10, Santa Cruz Technologies, Santa Cruz, CA., USA), a actinin (clone EA-53, Sigma Aldrich Ltd.) and a-smooth muscle actin (clone SPM332. Abeam Ltd.), using antibodies at 1 : 100-1 :250 dilution for 1 h at room temperature. After washing in PBS, appropriate Alexa-conjugated secondary antibodies (Invitrogen Ltd.) were applied at 1 : 1000 dilution for 1 h at room temperature. Cells were also stained with antibodies CD34-FITC (clone AC136, Miltenyi Biotec.) and CD31 (clone WM59, BD Biosciences) at 1 : 100 for 1 h at room temperature. Cells were imaged with a Nikon TE300 inverted fluorescent microscope (London, England) and images captured using Hamamatsu Photonics CCD camera (Welwyn Garden City, England) and processed with SimplePCI software (Digital Pixel, Brighton, England). To quantify percentages of different cells in this heterogeneous population, cells were analysed by FACS analysis. Following the cardiac differentiation
protocol in Example 2, day 14-21 iPS derived cells were harvested using 1 x accutase, pelleted at 300 g for 10 min, re-suspended in 100 μΙ PBS and fixed by 900 μΙ 4% (w/v) formaldehyde / PBS for 15 min on ice. After 3 washes with PBS, fixed cells were permeabilized using the Fixation/Permeabilization Kit (BD cytofix/Cytoperm™, BD Biosciences). The cells were stained for cardiac troponin I, troponin C, alpha-actinin and alpha-smooth muscle actin, using antibodies at 1 :20-1 :50 or CD34-FITC and CD31 -PE at 1 : 10, all for 1 h on ice. After washing, appropriate Alexa-conjugated secondary antibodies were applied at 1 : 1000 dilution for 1 h on ice.
[0049]EXAMPLE 5 - Generating eGFP positive iPS cells:
[0050] C18 iPS cells were transduced with lentivirus expressing eGFP at low moi (< 1 pfu/cell). At 48h post-transduction, eGFP positive iPS cells were harvested and plated at low density with colonies screened by fluorescence microscopy after 7-10 days. eGFP positive colonies were selected for further expansion, and the most highly expressing sub-clone was selected for further in-vivo studies.
[0051]EXAMPLE 6 - Rat myocardial infarction and cell administration:
[0052] The left anterior descending (LAD) coronary artery of female RNU-RNU rats (180-230 g) was occluded to induce ischemia reperfusion injury. In brief, following anaesthesia and thoracotamy, the pericardium was removed and a 5-0 prolene suture placed under the LAD, about 2 mm from the origin. The suture was tied around a small piece of PE tubing, occluding the LAD, and the chest closed. After 50 minutes, the chest was re-opened and the tubing removed to allow reperfusion. Ten minutes after reperfusion, iPS cells (2 x 106 in 50 μΙ HBSS medium; n = 4) or medium alone (50 μΙ; n = 5) were injected in the peri-infarct region. In sham animals (n = 3), the thoracotamy was performed but no stitch placed in the heart.
[0054] Cardiac cine MRI was performed as described (29) at 2 days and at 2, 6 and 10 weeks after infarction. Briefly, rats were anesthetized with 2.5% (v/v) isoflurane in O2, positioned supine in a purpose built cradle and lowered into a
60 mm birdcage coil in a vertical bore 500 MHz, 1 1 .7 T MR system with a Bruker console running Paravision 2.1 .1 . A stack of 7-8 contiguous 1 .5 mm true short axis ECG-gated cine images (field of view 51 .2 mm2, matrix size 256 x 256 zero filled to 512 x 512 giving a voxel size of 100 x 100 x 1500 pm, echo time/repetition time (TE/TR) 1 .43/4.6 ms, 17.5° pulse, 25-35 frames per cardiac cycle) were acquired to cover the entire left ventricle. The end diastolic and end systolic volumes were measured for each slice using Image J (at http://rsbinfonihgov/ij, N IH, Bethesda, Maryland, USA) and summed over the whole heart. Stroke volume was calculated as end diastolic volume minus end systolic volume. The ejection fraction was calculated as the stroke volume divided by the end diastolic volume. The akinetic area of the myocardial surface was calculated as the average of the endocardial and epicardial circumferential lengths of the thinned, akinetic region of all slices, measured at diastole, multiplied by the slice thickness, and expressed as a percentage of the total myocardial surface (30).
[0055]Evaluating iPS cell engraftment at 10 weeks post-MI:
[0056] Hearts were harvested 10 weeks post-surgery and fixed in 1 % (w/v) formaldehyde/PBS at 4°C overnight. Fixed tissues were paraffin-embedded for histology and sectioned at a thickness of 6-7 micrometers, transversely across the infarct zone. Sections were de-paraffinized and hydrated with deionised water. Immuno-fluorescence was carried out with anti-GFP (Invitrogen Ltd. ), anti-cardiac troponin I (clone EP1 106Y, Abeam Ltd.), anti-a- actinin (clone EA-53, Sigma Aldrich Ltd.) and anti-a-smooth muscle actin (clone SPM332, Abeam Ltd.) at 1 : 100-1 :400 dilution, and then appropriate Alexa-conjugated secondary antibodies (Invitrogen) applied at 1 : 1000 dilution. Slides were then treated with 0.1 % (w/v) sudan black in PBS for 10 min at room temperature with further washes in deionised water prior to visualisation. For immunohistochemistry, hydrated sections were incubated with 6% (v/v) H2O2 in methanol for 30 min at room temperature prior to staining with the relevant antibodies. Typically, anti-GFP and cardiac troponin I antibodies were applied as above and VECTASTAIN Elite ABC kit and DAB substrate kit for peroxidase added as described by the manufacturer (Vector laboratories, Orton Southgate, Peterborough, UK). Finally sections were stained with
hematoxylin (Sigma Aldrich Ltd.). All cells and slides were imaged with a Nikon ECLIPSE E600 microscope and images captured using a Nikon AxioCam and processed using PCISimple software as above. Statistics: Results are presented as means ± standard errors. Statistical assessment was conducted using analysis of variance at 10 weeks, with a post hoc test with Tukey correction, p values are presented for differences between infarct control group and iPS treatment group. For in vivo studies, n=5 for infarct control, n=4 for iPS treated and n=3 for sham operated groups.
[0057]EXAMPLE 7 - Inducing efficient cardiac differentiation strategies from human iPS cells as monolayers.
[0058] Whilst several differentiation strategies exist for the derivation of cardiovascular cells from ES and iPS cells, both for mouse and human cells (27, 31 ) these usually require the formation of embryoid bodies or the use of serum. Some also describe cardiac differentiation as monolayers (10, 15), and others have described differentiation of specific cells types, such as endothelium, from pluripotent stem cells using signal inhibition (32).
[0059]In this novel protocol, human iPS cell monolayers were exposed to reduced activin A (50 ng/ml) in StemPro34 basal media for a short period of time (4 h) to prevent the extensive cell death that occurred after exposure to high doses of activin A (100 ng/ml) for extended periods of up to 24 hours (data not shown).
[0060] Additional modifications included continuous exposure to lower levels of activin A (5 ng/ml), bFGF (5 ng/ml) and BMP4 (10 ng/ml) for a further 92 h, followed by culture in StemPro34 for 10 days with no further supplementation. Figure 1 a outlines this optimized protocol with bright field photos of cells at each significant time point. At dO, iPS cells were clearly evident, with large flat colonies, and cells with a large nucleus and scant cytoplasm. By 4h incubation with Activin A in StemPro34, although there was extensive cell death, a majority of live cells remained attached (see Fig 2a day 2) and expanded over the early stages of culture, and continued to proliferate until sheets of 'stromal like' cells were overlaid by cardiac progenitors. These
overlying cells fused as single structures that eventually beat with other neighbouring structures, often as a single sheet.
[0061]When human iPS cells are differentiated as embryoid bodies using directed approaches such as those described by Yang et al. , typically less than 5% of the embryoid masses are seen beating (unpublished data) and approaches described by Laflamme et al. (10) result in such extensive cell death that very few iPS cells survive for subsequent analysis. In contrast the presently defined strategy provides for the efficient differentiation of human iPS cells as monolayers towards the cardiac lineage. Fig. 1 b shows a representative analysis of the cell surface expression of biomarkers, over days 4 to 8 of culture, that are associated with the cardiovascular lineage.
[0062]This shows the formation of PDGFalphaR positive populations at day 5, which subsequently increased in numbers over 4 to 8 days (Figure 1 b) and which represented up to 38.7% ± 8.2% of cells on day 6. This marker has previously been associated with the mesoderm (33), and more specifically the cardiac lineage (28, 31 , 34) whilst it has been shown recently to be induced by factors such as Mespl (35), responsible for mesodermal fate specification.
[0063]The cells also show staining for Tie2, a marker thought to identify the endothelium (36) (as well as other cells), and which with KDR positive cells, represented 5.4% ± 2.9% of cells on day 8. This, upon co-staining for PDGFaR, appeared to be a distinct population that was Tie2+KDR+PDGFaR".
[0064]It is unclear at this time whether these cells arise from the cardiac progenitor described by Yang and colleagues (27) or from a distinct progenitor that can form independently during differentiation, such as the hemangioblast (37).
[0065]Fig. 1 c, shows consequent loss of ES specific markers SSEA3 and TRA-1 - 80. Human ES and iPS cells should be dual positive for these markers (see
dO), suggesting that iPS cells do not persist at day 6 of the cardiac differentiation protocol.
[0066]When these differentiating monolayers were assessed by immuno- histochemistry (Figure 2), they were highly positive for cardiac markers, troponin I, alpha-actinin and SMA. In the case of alpha-actinin, staining clearly showed sarcomeric Z lines with highly distinctive striations characteristic of cardiomyocytes.
[0067] Furthermore, strong co-staining was observed for CD34 and CD31 , suggestive of an endothelial cell type. Although these markers are not specific to endothelium, and are expressed on hematopoietic cells and a subset of mesenchymal stem/stromal cells, the CD34+ cells were negative for CD45 and expressed low/negligible CD90, which is data that support an endothelial phenotype. Interestingly, these iPS-derived endothelial cells formed tubule networks that were shown to be integrated into regions that were rich in cardiomyocytes and smooth muscle, but represented just 1 .66% of the total population when assessed by FACS (see Figure 2).
[0068] Upon subsequent assessment of day 14 cultures by FACS analysis, positive staining for differentiated cardiac cell markers such as troponin I, a-actinin, a- smooth muscle actin (SMA) and CD34 was quantitatively assessed (Figure
[0069]2g). 39.8% ± 15.1 % (n = 5 independent cultures) of the population was positive for troponin I, thus representing cardiomyocytes, whilst cells expressing a-actinin correlated closely at 36.1 % ± 2.5%.
[0070] SMA, a marker for smooth muscle, was detected on 6.4% ± 3.6% of cells analysed, and expression of the endothelial marker CD31 was 1 .66% ± 0.75% of cells analysed.
[0071] These data match the FACS profiling for cardiovascular markers over days 4- 8, and suggest that the present protocols favour the formation of cardiomyocytes and smooth muscle, with endothelium forming to a lesser extent. The high level of cardiomyocyte formation in the monolayer gave rise to extensive beating masses that resulted in sheets of contractile tissue.
[0072]The present protocol for cardiac differentiation of iPS cells and production of contractile cardiomyocyte from monolayers is highly reproducible and
therefore reliable, and to-date represents the most efficient and straight forward approach for generating such cells.
[0073]EXAMPLE 8 - Functional assessment of human iPS cell derived cardiac progenitors in a rat model of ischaemia-reperfusion injury:
[0074] Following reperfusion, 2 x 1 06 day 6 eGFP positive, cardiac differentiated iPS cells were administered by intra-myocardial injection. Cardiac function, measured at 2 days using in vivo cine-MRI (as shown in Fig. 3A and quantified in Fig.3B), showed no significant difference in ejection fraction, end systolic volume or end diastolic volume between control infarcted and iPS- treated rat hearts.
[0075] Although there were no significant differences in cardiac morphology and function between infarcted and iPS-treated hearts at any time point, hearts injected with iPS-derived cardiac differentiated cells showed attenuated functional decline after infarction (62 ± 4%) when compared to infarct control (45 ± 9%), at 10 weeks. Although the ejection fraction was not significantly different from those of the infarct control animals at 10 weeks there was a non-significant trend (p<0.2 ). This was also true for end systolic volume (ESV) (p=0.2) and average LV mass (p<0.2) which further suggests that the protection from decline in heart function observed after myocardial infarction, was conferred by introduction of iPS derived cardiomyocyte.
[0076]Although the protective effects from injection of the iPS-derived cardiac cells were generally modest after myocardial infarction, the functional data showed a non-significant trend toward an effect in the iPS group not seen in other long-term functional studies (4).
[0077]EXAMPLE 9 - Histological assessment to determine retention of eGFP positive cells at 10 weeks post-transplant:
[0078] To demonstrate that protection of rat hearts from AMI resulted from the injected human iPS derived cardiac cells, the inventors showed that they had engrafted into the rat heart, and were retained for up to 10 weeks post myocardial infarction Fig 4. At this time hearts were recovered, preserved by fixation, and sectioned and stained for cardiac specific markers. Although large sheets of engrafted tissue were not evident, areas around the infarct
zone that retained eGFP positive cells that were clearly of the cardiac lineage were shown, since they were dual positive for eGFP and alpha-actinin, SMA or troponin I, when assessed by immuno-fluorescence (Figure 4). Some heterogeneity in signal is observed in Fig. 4a, since silencing of the eGFP transgene was experienced (approx. 58% of iPS cells were detectably eGFP positive, whilst the remaining cells were silenced), so that in vivo, some cells appeared positive for human specific Troponin I only, and not GFP (hence staining red only). Conversely not all iPS-derived cardiac differentiated cells formed cardiomyocytes and so these did not stain for Troponin I, and thus may have appeared green only. However, a large number of cells with dual staining for both eGFP and Troponin I was observed as expected. Similar staining patterns were observed for D-actinin (Fig. 4b) and smooth muscle actin (Fig. 4c) in those hearts treated with eGFP-positive iPS cells (lower panels). eGFP positive cells were counted to be at 22.04% (SD ± 3.4% n=3), whilst Troponin I positive cells were counted to be 9.38% (SD ± 0.01 %, n=3), in areas around the infarct zone.
[0079]Control infarct only tissue (upper panels in Figure 4) showed no expression of human specific Troponin I or eGFP, however alpha-actinin and SMA expression was detected since antibodies used in these groups were not specific for the human antigen. In these control panels there was no eGFP expression and thus no co-localization. From these studies, it is suggested that retention of a limited number of cells at 10 weeks is highly significant, since engrafted tissue is lost over this time, even in immuno-compromised models. To observe the presence of transplanted cells at 10 weeks indicates that many more iPS-derived cardiac differentiated cells were retained and engrafted during the initial injection and recovery from infarct, resulting in the significant benefit in cardiac function observed with the iPS treated group, compared to the infarct group, over subsequent weeks.
EXAMPLE 10- Demonstration that cardiac progenitors possess a haematopoietic potential via a haemogenic endothelium that gives rise to erythroid and myeloid lineages.
[0080]When cardiac differentiating cultures are exposed to VEGF from day4-day8, we show that cells positive for CD31 + CD45+ are induced (Fig 5a to c), which typically arise from a haemogenic endothelium. These cells when expanded in haematopoietic culture conditions yield myeloid cells (CD14+) (Fig 5d-f) in methocult, or red blood cell (CD235+) cells (Fig 5g) in cardiac culture supplemented with erythroid differentiation factors. Other potential lineages include megakaryocyte and lymphoid cells. Although these were not tested, it is entirely feasible that these cells may from this method, given that hematopoietic cells arising from haemogenic endothelium in animal models is known to give rise to the haematopoietic stem cell.
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