WO2014070113A1

WO2014070113A1 - In situ cardiac tissue engineering

Info

Publication number: WO2014070113A1
Application number: PCT/SG2013/000467
Authority: WO
Inventors: Eliana Cecilia MARTINEZ VALENCIA
Original assignee: National University Of Singapore
Priority date: 2012-11-02
Filing date: 2013-10-30
Publication date: 2014-05-08
Also published as: GB201219707D0

Abstract

The disclosure relates to the preparation of cardiac tissue grafts in situ for inducing post-ischemic vascularisation and recovering cardiac function of damaged myocardium. It also provides methods for the treatment of heart disease, in particular ischemic heart disease.

Description

In Situ Cardiac Tissue Engineering

Field of the Invention

The disclosure relates to the provision of cardiac tissue grafts for inducing post-ischemic vascularisation and recovering cardiac function of damaged myocardium. It also provides methods for the treatment of heart disease, in particular ischemic heart disease.

Background to the Invention

The term "stem cell" represents a generic group of undifferentiated cells that possess the capacity for self-renewal while retaining varying potentials to form differentiated cells and tissues. Stem cells can be pluripotent or multipotent. A pluripotent stem cell is a cell that has the ability to form all tissues found in an intact organism although the pluripotent stem cell cannot form an intact organism. Furthermore, it is known that human somatic cells can be re-programmed to an undifferentiated state similar to an embryonic stem cell. For example, WO2007/069666 describes re-programming of differentiated cells (e.g. mouse fibroblast cells) without the need to use embryonic stem cells.. Nuclear re-programming is achieved by transfection of retroviral vectors into somatic cells that encode nuclear re-programming factors, for example Oct family, Sox family, lf family and M c family of transcription factors. The somatic cells de-differentiate and express markers of human embryonic stem cells to produce an "induced pluripotent cell" [iPS]. In Takahashi ef a/ [Cell vol 131 , p861-872, 2007] adult human dermal fibroblasts with the four transcription factors: Oct3/4, Sox2, Klf4, and c- Myc de-differentiate to human ES cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes and telomerase activity.

A multipotent cell has a restricted ability to form differentiated cells and tissues. Typically, adult stem cells are multipotent stem cells and are the precursor stem cells or lineage restricted stem cells that have the ability to form some cells or tissues and replenish senescing or damaged cells/tissues. Generally they cannot form all tissues found in an organism, although some reports have claimed a greater potential for such 'adult' stem cells than originally thought. Examples of multipotent stem cells include mesenchymal stem cells. Mesenchymal stem cells differentiate into a variety of cell types that include osteoblasts, chondrocytes, myocytes, adipocytes and neurones. Typically, mesenchymal stem cells are obtained from bone marrow. Currently, stem cell therapies are exploring different sources of pluripotent and multipotent stem cells and cell culture conditions to efficiently differentiate stem cells into cells and tissues suitable for use in tissue repair. The heart is an organ that functions to circulate oxygenated blood to the major organs of the body and deoxygenated blood carrying carbon dioxide to the lungs. In addition to the supply of oxygenated blood to the major organs the heart is supplied with oxygen via the coronary arteries. The heart comprises cardiac muscle which forms the walls of the heart and is an involuntary striated muscle which is adapted to be resistant to fatigue. Failure to supply sufficient oxygen to essential organs results in a condition called ischemia which usually is caused by a critical coronary artery obstruction, but can be irreversible. Myocardial ischemia is a condition that can be asymptomatic until such time as the supply of oxygen to cardiac muscle becomes restricted to the extent that the muscle fails and a heart attack occurs.

Ischemic heart diseases (IHD) are globally the primary cause of deaths. An estimated 17.3 million people died from cardiovascular diseases in 2008, representing 30% of all global deaths. Of these deaths, an estimated 7.3 million were due to coronary heart disease and 6.2 million were due to stroke. These figures are likely to increase in future, placing a huge financial burden on the health care system.

IHD are characterised by reduced blood supply to the heart muscle, causing infarction and cell death of the affected area. Heart muscle cells lose after birth their capacity to divide prohibiting the self-regeneration of the heart after injury. Upon myocardial infarction, scar tissue develops over the damaged regions reducing the contractile function of the heart, leading to ventricle wall thinning and ultimately heart failure.

Treatment of damaged myocardium requires replacement of the scar tissue with functioning cardiac muscle tissue. However, despite recent advancements in the area of cell tissue engineering, success rates are low and the requirements for developing functioning myocardial tissue are meticulous. Selection of the right cell population, the establishment of scaffold-like structures mimicking native tissue matrices, construction of tissue patches of desired size and enabling vascularizations are detrimental factors for engineering functional tissue grafts.

Cell based therapy includes the administration of cells to the diseased heart in order to reestablish, at the desired location, a structurally and functionally intact unit. Transplanted cells are required to differentiate, proliferate and induce vasculogenesis. For that purpose many different cell types including skeletal myoblasts, cardiac stem cells, adipose stem cells, bone marrow-derived hematopoiec and mesenchymal adult stem cells, or pluripotent embryonic cells have been trailed with various success rates. However, a major drawback using cell based therapies is the low cell survival and limited engraftment when delivering cell suspensions. Improvements were observed when cells were transplanted forming a cardiac-like tissue structure, which is promoted by seeding cells onto a porous, fibrous or hydrogel scaffold. US7, 396, 537 discloses a composition of a cardiac patch comprising different layers using collagen hydrogel supported by an intermediate scaffolding layer formed of biodegradable co-polymers of glycolic and/or lactic acid attached to a reinforcement layer of non-biodegradable support material. Others disclose devices made of support scaffolds and cell sheets; see US2006/0153815. Mechanical stability and elasticity was addressed in US2004/017712 by developing a tubular artificial tissue scaffold of high elasticity having aligned biofibrils seeded with cells. Besides using scaffolds, techniques have also been developed to engineer scaffold free tissues, composed only of cells and the matrix they secrete, circumventing common problems occurring when using scaffolds, as residual polymer fragments can interfere with the cell organisation and an inherent weakness of tissue engineered vessel.

Vascularisation is a major bottleneck as tissue grafts require oxygenation for survival. The preparation of a cardiac patch with a large surface area, perfect cellular organisation and optimal vascularisation remains a major challenge. Different scaffold types are known to support vascularisation and the addition of oxygen carriers, pro-angiogenic factors such as VEGF, or the use of engineered cells expressing pro-angiogenic factors have shown to increase vascularisation; for example WO2006/121532 discloses the transplantation of cells engineered to express one or more pro-angiogenic factors, and moreover express anti- apoptotic factors to increase cell life.

The present disclosure relates to cardiac tissue engineering as exemplified in a non-limiting manner by a novel cardiac tissue graft combining a scaffold-free generated tissue of sub- amniotic cord-lining mesenchymal stem cells (CL-MSC) coated with human umbilical vein endothelial cells (HUVEC) embedded within a fibrin scaffold providing controlled and homogeneous cell delivery onto the target area in a clinically-relevant time-scale. Transplantation of the 3D tissue graft results in improved cardiac function and myocardial revascularisation. Statements of Invention

According to an aspect of the invention there is provided a therapeutic cell composition comprising: a spheroid comprising lineage restricted stem cells derived from pluripotent stem cells or multipotent stem cells and activated endothelial cells wherein the combined cell preparation is associated with a biodegradable cell support matrix.

In a preferred embodiment of the invention said lineage restricted pluripotent stem cells are derived from induced pluripotent stem cells.

In an alternative preferred embodiment of the invention said lineage restricted stem cells are derived from embryonic stem cells; preferably human embryonic stem cells.

In a preferred embodiment of the invention said multipotent stem cells are cardiac stem or progenitor cells.

In a preferred embodiment of the invention said multipotent stem cells are mesenchymal stem cells. Preferably said mesenchymal stem cells are selected from the group consisting of: bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord blood mesenchymal stem cells or sub-amniotic cord-lining mesenchymal stem cells.

Preferably said stem cells are angiogenic stem cells.

In a preferred embodiment of the invention said angiogenic mesenchymal stem cells are sub-amniotic cord-lining mesenchymal stem cells.

In a preferred embodiment of the invention said activated endothelial cells are umbilical vein endothelial cells.

"Activated endothelial cells" are endothelial cells that express pro-angiogenic factors such as VEGF and therefore have the capacity to promote angiogenesis of associated cells.

Preferably, said mesenchymal stem cells are mouse, rat, primate or human.

In a preferred embodiment of the invention said cell support matrix polymerises at body temperature.

Preferably the cell support matrix comprises natural or synthetic polymers.

In a preferred embodiment of the invention said cell support matrix comprises a hydrogel.

In a preferred embodiment of the invention said cell support matrix comprises or consists essentially of fibrin. According to a further aspect of the invention there is provided a cell composition according to the invention for use in the treatment of heart disease.

In a preferred embodiment of the invention heart disease is selected from the group: ischemic heart disease, coronary heart disease, congestive heart failure, cardiomyopathy or myocardial infarction.

In a preferred embodiment of the invention said heart disease is ischemic heart disease.

In a preferred embodiment of the invention said stem cells and/or said activated endothelial cells are modified.

"Modified" means obtaining naturally occurring stem cells or endothelial cells and altering either the genome by genetic modification or by the addition of biologically active molecules into or on the cells to alter the biological properties of the stem/endothelial cells. For example, cells can be modified by transfection of nucleic acid molecules, [e.g. gene therapy vectors], encoding factors that enhance the biological activity of the cells. This can be achieved by expression of pro-angiogenic factors or by expression of factors that modulate the expression of pro-angiogenic factors. Within the scope of the invention are pro- angiogenic factors such as, for example VEGF-A, B, C and D. Other examples include microRNAs. MicroRNAs [are small 21-23nt] single stranded RNAs that are processed from longer precursor RNAs encoded by the genome of an organism and are wholly or partially complementary to mRNAs expressed by the organism and have the function to down regulate expression of genes that encode the mRNAs. Mechanistically miRNAs function in the same way as siRNA and use essentially the same enzymatic machinery.

According to a further aspect of the invention there is provided a surgical procedure to repair heart tissue in a subject in need of heart surgery comprising the steps: i) providing a cell composition according to the invention;

ii) preparing a incision in the thoracic region of the subject thereby exposing the heart;

iii) contacting the heart tissue in need of repair with the composition of (i) above; and optionally;

iv) repeat step (iii).

In a preferred method of the invention said surgical procedure is video-assisted thoracoscopic surgery. In a preferred method of the invention the cell composition is applied to the heart tissue via a catheter wherein the combined cell preparation is not fully polymerized.

A catheter is one means by which the combined cell preparation may be applied. Alternatively a thoracoscopic device could also be used for this purpose.

In a preferred method of the invention said subject is human.

In a preferred method of the invention the repaired heart tissue is a result of a heart disease selected from the group: ischemic heart disease, coronary heart disease, congestive heart failure, cardiomyopathy or myocardial infarction.

The cell composition according to the invention is administered in effective amounts. An "effective amount" is that amount of the cell composition that alone, or together with further doses, produces the desired response. In the case of treating a particular heart disease the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any) and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

When treating myocardial ischemia the desired response is the symptomatic treatment of the consequences of the disease. This may involve only the partial improvement of the symptomatic consequences of the disease, although more preferably, it involves complete improvement of the symptomatic consequences of the disease. This can be monitored by routine methods. More particularly improvements in myocardial ischemia can be monitored by any one of the following indicia: tolerance to exercise and physical stress; exercise stress ECG testing; heart function on echocardiography or ECG signs of ischemia and cardiac MRI to provide details of cardiac structure. According to a further aspect of the invention there is provided a method for the preparation of angiogenic spheroids comprising lineage restricted stem cells derived from pluripotent stem cells or multipotent stem cells comprising the steps: i) providing a growing culture of stem cells in cell culture medium;

ii) providing hypoxic cell culture conditions sufficient to induce expression of pro- angiogenic factors by said stem cells; and optionally

ii ) contacting the induced angiogenic stem cells with a preparation comprising activated endothelial cells to form a combined cell preparation.

In a preferred method of the invention said lineage restricted stem cells are derived from induced pluripotent stem cells.

In an alternative preferred method of the invention said lineage restricted stem cells are derived from embryonic stem cells; preferably human embryonic stems.

In an alternative preferred method of the invention said multipotent stem cells are cardiac stem or progenitor cells.

In a preferred method of then invention said stem cells are mesenchymal stem cells.

Preferably said mesenchymal stem cells are selected from the group consisting of: bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord blood mesenchymal stem cells or sub-amniotic cord-lining mesenchymal stem cells.

In a preferred method of the invention said mesenchymal stem cells are sub-amniotic cord- lining mesenchymal stem cells.

In a preferred method of the invention said combined cell preparation is associated with a biodegradable cell support matrix.

In a preferred method of the invention said cell support matrix comprises or consists essentially of fibrin.

In a preferred method of the invention said hypoxic conditions to induce expression of pro- angiogenic factors is a hanging drop cell culture.

In a preferred method of the invention angiogenic spheroids are harvested when their diameters reach about 250-30Όμιτι.

In a preferred method of the invention angiogenic spheroids are cultured for up to about 3 days prior to combining with activated endothelial cells. Preferably the combined cell preparation is cultured for up to about 4 days.

A "hanging drop culture" is known in the art and provides a means to grow cells, for example stem cells, in dimensions other than monolayer culture. Drop cultures are formed using cells and growth medium by forming droplets comprising the cells to minimize the surface area to volume thereby reducing evaporation and creating low oxygen tension. The drops are suspended from the surface of a cell culture vessel. Mesenchymal stem cells according to the invention develop into spheroid structures within the drop culture which because of the hypoxic conditions express pro-angiogenic factors and thereby provide an angiogenic mesenchymal spheroid which when combined with activated endothelial cells provides a superior graft composition for use in the method of the invention.

According to a further aspect of the invention there is provided a kit comprising: i) lineage restricted stem cells derived from pluripotent stem cells or multipotent stem cells;

ii) activated endothelial cells;

iii) a biodegradable cell support matrix; and optionally

iv) cell growth medium for the maintenance of stem cells and/or activated endothelial cells.

In a preferred embodiment of the invention said lineage restricted stem cells are derived from induced pluripotent stem cells.

In an alternative preferred embodiment of the invention said lineage restricted stem cells are derived from embryonic stem cells; preferably human embryonic stems.

In an alternative preferred embodiment of the invention said multipotent stem cells are cardiac stem or progenitor cells.

In a preferred embodiment of then invention said stem cells are mesenchymal stem cells.

In a preferred embodiment of the invention said kit includes sub-amniotic cord-lining mesenchymal stem cells.

In a further embodiment of the invention said kit includes a biodegradable support matrix comprising fibrin. In a preferred embodiment of the invention said kit further comprise products used in the surgical procedure according to the invention.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. "Consisting essentially" means having the essential integers but including integers which do not materially affect the function of the essential integers.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where, the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following figures:

Figure 1 : Method for subamnion-CL-MSC angiogenic spheroid-enriched grafts delivery through lateral thoracotomy (SASG) (A), or by^" minimally invasive video-assisted thoracoscopic surgery (SASG-VATS) (B-G). (A) Epicardial implantation of 3D graft onto the left ventricular (LV) scar area. Inset showing pre-made fibrin graft containing angiogenic spheroids. (B) For delivery of spheroids & fibrin to form an epicardial patch in situ, a 2mm straight forward telescope was inserted through a 3mm incision at the 8^th intercostal space, mid-axillar line. (C) A fibrin/DPBS and spheroids mixture was loaded into a 16G catheter using a 1 ml syringe, which was then inserted into the thorax through an incision made at the 4^th intercostal space, parasternal line. (D) Surgical wounds after VATS procedure. Note small wounds size compared to previous thoracotomy. (E-G) The partially gelled fibrin/DPBS/spheroids mixture was deposited epicardially onto the scar area under video- thoracoscopic visualization. The mixture was then allowed to polymerize completely before the next batch was applied. This process was repeated 3 times until the scar area was totally covered with spheroids. (H-J) The same procedure (without spheroids) was done for fibrin controls (FG-VATS). CL-MSC, cord-lining mesenchymal stem cells; Figure 2: ln-vivo and ex-vivo bioluminescence imaging (BLI) following SASG and SASG- VATS implantation. Longitudinal evaluation of donor cell viability in-vivo during the first 2 weeks after (A) SASG and (B) SASG-VATS implantation. In-vivo donor CL- SC-G FP-Fluc viability was comparable in both SASG and SASG-VATS groups throughout the study. A non-significant reduction in cell viability was detected during the first week after graft implantation, followed by a significant decrease in bioluminescence from day 1 to 14 in both groups (^**P<0.01 and ^***P<0.001) (C). Ex-vivo BLI 4 weeks after treatment revealed surviving donor CL-MSC in the left ventricular scar area from both groups (right panels in A & B), while no difference was detected in long-term donor CL-MSC survival between SASG and SASG-VATS treatment (D). CL-MSC, cord-lining mesenchymal stem cells; GFP, green fluorescent protein; flue, firefly Iuciferase; SD, standard deviation. (E) Echocardiography comparison of left ventricular (LV) remodelling and function between infarcted untreated rats (Ml), fibrin graft- (FG), SASG, fibrin graft generated in situ via VATS- (FG-VATS), and SASG-VATS- treated rats, 6 weeks after myocardial injury. SASG and SASG-VATS therapy attenuated LV adverse remodelling and preserved cardiac function 6 weeks post-injury (i.e. 4 weeks after treatment). Treated hearts displayed more conserved LV dimensions when compared to Ml, both in systole and diastole, whereas significant enhancement in ejection fraction was found in SASG and SASG-VATS animals compared to Ml and in SASG compared to FG. Furthermore, higher fractional shortening was observed in both treatment groups compared to Ml and their respective FG controls. Significance in group comparisons vs. Ml is indicated as follows: ^*P<0.05; *^*P<0.01; ^#P<0.001 ; **P<0.0001. (F) Morphometry studies of explanted hearts 4 weeks after treatment. Representative mid-ventricular cross- section of Masson's trichrome-stained sections (40x) showing more conserved left ventricular (LV) dimensions and LV wall thickness, as well as less scar tissue in SASG and SASG-VATS. Scale bars, 1000 pm. (G) Measurement of the percentage of LV containing fibrosis (calculated by dividing the midline length of the infarcted LV wall by the midline length of total LV wall) [39] revealed that treatment with SASG and SASG-VATS led to smaller LV scar size compared to the other groups. ^**P<0.01 vs. Ml; ^**^**P<0.0001 vs. Ml. SD, standard deviation.

Figure 3: Representative micrographs of the left ventricular scar area stained with rat endothelial cell antigen-1 (RECA-1⁺) to visualize host blood vessels in failing rat hearts treated with (A) subamnion-CL-MSC angiogenic spheroid-enriched grafts implanted through lateral thoracotomy (SASG); (B) fibrin grafts implanted by thoracotomy (FG); (C) SASG implanted by minimally invasive video-assisted thoracoscopic surgery (SASG-VATS); (D) fibrin grafts implanted though VATS (FG-VATS); or in (E) untreated (Ml) rats, 4 weeks after treatment; (200x). RECA-1⁺ blood vessels (red), nuclei (DAPI, blue). Scale bar indicates 50 μιη. (F) The LV scar area in SASG and SASG-VATS contains a rich network of blood vessels. The number of RECA-1⁺ blood vessels in the LV scar area per 200x field was higher in SASG-treated hearts than FG (P<0.001 ) and Ml hearts (****P<0.0001 ). Similarly, SASG-VATS hearts had more vasculature than FG-VATS (P<0.01), and Ml (**P<0.01). Host RECA-1⁺ vasculature were also observed within the graft area of SASG (G) and SASG- VATS (H). Confocal micrographs of the epicardial graft and LV scar area after perfusion with 1 ,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) to visualize functional blood vessels in failing rat hearts treated with SASG and SASG-VATS (l-K). Merged xy confocal image with the transmitted light channel of the epicardial graft area showing the presence of functional blood vessels throughout the implanted graft (100x) (I). Inset of image (200x and 400x) correspond to 3D reconstruction of z-stacks from the same area showing blood vessel networks surrounding and penetrating the extracellular matrix (ECM) of a spheroid embedded in the graft. Abundant sprouting of neo-vessels was seen within the spheroid's ECM (arrows). Dotted line indicates host/graft interface. Right panel in (I) shows xy of the inset merged with the transmitted light channel and DAPI (in blue). 3D images of angiogenic sprouts and pseudopodial processes at a viewing angle of 0° in the LV scar area of SASG- (J) and SASG-VATS- treated hearts (K). Left panel corresponds to merged xy confocal image with the transmitted light channel, whereas right panels correspond to 3D reconstruction of z-stacks from the same area. Arrows in (J-K) indicate angiogenic sprouts in the LV ischemic area. CL-MSC, cord-lining mesenchymal stem cells; SD, standard deviation;

Figure 4: Masson's trichrome-stained heart sections of (A) untreated Ml hearts, as well as (B) FG-, (C) SASG-, (D) FG-VATS- and (E) SASG-VATS-treated hearts 4 weeks after treatment (400x) showing more vascularity in SASG/SASG-VATS groups. Arrows indicate arterioles; scale bar, 20 im. The left ventricular (LV) wall of SASG (F) and SASG-VATS (G) had abundant blood vessels containing red blood cells (100x). Arrowheads indicate epicardial graft's remnants. Higher blood vessel density (arterioles and capillaries) was found in the LV infarct area (IA) of SASG/SASG-VATS hearts (H). Increased arteriole counts were also found in the LV border zone (BZ) of SASG/SASG-VATS rats (I). *P<0.05 vs. Ml, **P<0.01 vs. Ml; ***P<0.001 vs. Ml. ^#P<0.0001 vs. Ml. Rich vascularity was also observed within the graft of SASG (F & J) and SASG-VATS (G & K), infiltrating the fibrin matrix and the spheroids' ECM (asterisks). Scale bar in J & K, 100 μητ SD, standard deviation; hpf, high-power field;

Figure 5: Confocal micrographs of hearts that received treatment with subamnion-CL-MSC angiogenic spheroid-enriched grafts implanted through lateral thoracotomy (SASG) (A,C,E), or by minimally invasive video-assisted thoracoscopic surgery (SASG-VATS) (B, D,F), after 4 weeks of treatment showing alpha-smooth muscle actin positive (a-SMA⁺) blood vessels and donor CL-MSC-GFP⁺. CL-MSC-GFP⁺ were observed within the spheroids or within the graft in close relationship with arterioles, which tended to surround and infiltrate the spheroids (A-B). Yet, some of the delivered human CL-MSC contributed to vascularization, as GFP-expressing cells were colocalized with a-SMA⁺ cells in the LV scar (C-D) and graft area (E-F) from both groups, implying CL-MSC smooth muscle. Top panel shows merged image, whereas small panel below shows each staining: a-SMA⁺ blood vessels (red), CL- MSC-GFP⁺ cells (green), nuclei (DAPI, blue). CL-MSC, cord-lining mesenchymal stem cells;

Figure 6: Characterization of human cord-lining mesenchymal stem cells (CL-MSC). (A) Oil Red O staining Alcian blue, and Von Kossa, (100x) and demonstrating CL-MSC-GFP-fluc multi-lineage differentiation potential. (B-C) Surface antigen expression of CL-MSC-GFP- fluc as analyzed by flow cytometry. All markers were expressed in a cellular percentage (B) Grey histogram represents the isotype control for CD73, CD90 and CD105. (C) Blue histograms represent the antigen whereas red histograms correspond to the isotype control. GFP, green fluorescent protein; flue, firefly luciferase. CL-MSC, cord-lining mesenchymal stem cells;

Figure 7: A. Spheroids after 7 days of gravity-enforced culture. Compact and well delimited spheroids (250-30ΌμΓη diameter) were formed, in which cells were not discriminable by light microscopy. B. Scanning electron microscopy of CL-MSC-GFP-fluc and HUVEC angiogenic spheroids after 7 days in hanging drops. Low-magnification image (250x) shows compact spheroids with extensive extracellular matrix that only allows a few individual cells distinguishable on the surface. High-magnification image of inset (2,000x) shows cell-cell tight junctions (arrows). C-D. Western Blot analysis of VEGF protein level in monolayer cultures (HDFa, HUVEC and CL-MSC) and in uncoated CL-MSC spheroids after 3 days in hanging drop culture (CL-MSC Sph-D3), or coated spheroids after 5 (Coated Sph-D5) and 7 days in culture (Coated Sph-D7). Expression of VEGF monomers was comparable between HUVEC and CL-MSC- GFP-fluc cultured in monolayers. After assembly of CL-MSC- GFP- fluc in 3D spheroids, VEGF monomers were reduced and VEGF dimers increased compared to both CL-MSC and HUVEC monolayer cultures (***P<0.001 ). E. H&E staining showing distribution of angiogenic spheroids within fibrin grafts (SASG). F. Masson's trichrome staining of SASG revealed production of extracellular matrix (in blue) within the spheroids. G-H. Bioluminescence imaging of SASG at 1 , 3 and 7 days under static culture showed nonsignificant changes in donor CL-MSC- GFP-fluc viability. VEGF, vascular endothelial growth factor; HDFa, human dermal fibroblasts adult; HUVEC, human umbilical vein endothelial cells; CL-MSC human cord-lining mesenchymal stem cells; GFP, green fluorescent protein; flue, firefly luciferase; SASG, . subamnion-cord-lining mesenchymal stem cell angiogenic spheroid-enriched grafts; SD, standard deviation.

Figure 8: Apoptosis assessment within subamnion-CL-MSC angiogenic spheroid-enriched grafts in vitro. Representative confocal micrographs of active caspase-3 staining (red) in CL- MSC-GFP-fluc HUVEC spheroids embedded in fibrin grafts after 1 (A) , 3 (B) ,and 7(C) days in culture; (100x). Arrows are pointing, at some caspase 3-positive cells. CL-MSC-GFF^* (in green), DAPI⁺ nuclei (in blue); scale bars indicate 100 pm. (D) The percentage of apoptotic cells within the spheroids was significant at 3 and 7 days in culture, relative to day 1. (E) Determination of cell number (DAPI⁺ cells) within spheroids revealed that cell number within spheroids during SASG static culture from day 1 to 7 remained stable. CL-MSC, cord-lining mesenchymal stem cells. GFP, green fluorescent protein; flue, firefly luciferase; HUVEC, human umbilical vein endothelial cells; SD, standard deviation;

Figure 9: Confocal micrographs of subamnion-CL-MSC angiogenic spheroid-enriched grafts (100x) at 1 (A), 3 (B) and 7 days (C) in vitro, stained for human CD31. (A-C) Donor CL-MSC- GFP-fluc (in green) adopted a MSC in v/Vo-like elongated shape inside the spheroids and organized into compact cellular networks. HUVEC (in red) were found in close contact with CL-MSC at all time-points in static culture and assembled into branches capillary-like structures (arrows) while penetrating the spheroid's core [52]. (C) By day 7 of SASG static culture, HUVEC displayed sprouting (arrowheads), whereas some CL-MSC started to migrate outside of the spheroid into the fibrin matrix (asterisk). Scale bar, 100μηι. CL-MSC, cord-lining mesenchymal stem cells. GFP, green fluorescent protein; flue, firefly luciferase; HUVEC, human umbilical vein endothelial cells;

Figure 10: Alpha-smooth muscle actin expression in the native myocardium. Representative confocal micrographs of (A,F) untreated rat hearts (Ml), and those treated with (B,G) fibrin graft through thoracotomy (FG), (C,H) subamnion angiogenic spheroid- enriched grafts implanted through lateral thoracotomy (SASG), (D,l) FG implanted by minimally invasive video-assisted thoracoscopic surgery (FG-VATS), and (E,J) SASG delivered through VATS (SASG-VATS), after 6 weeks of myocardial injury. ct-SMA⁺ blood vessels are labeled in red, donor GFP⁺ cells in green and DAPI⁺ nuclei in blue. Upper panel (A-E) corresponds to xy confocal micrographs merged with the transmitted light channel to identify underlying left ventricular (LV) scar and graft areas (40x); white arrowheads pointing out at spheroids embedded within fibrin graft. Lower panels (F-J) correspond to micrographs taken in the LV scar area (200x). Abundant a-SMA⁺ blood vessels were found within the scar area of SASG and SASG-VATS groups compared to Ml and their respective FG/FG- VATS controls. Also, arterioles were seen infiltrating the fibrin grafts and surrounding spheroids. Scale bar indicates 100 μιη. Donor CL-MSC-GFP⁺ cells within the epicardial graft and left ventricular (LV) scar area of SASG (K, L & ) and SASG-VATS (M & O). CL-MSC- GFP⁺ (in white) were found either within the spheroids extracellular matrix or fibrin graft (black arrows). GFP⁺ cells within the graft area were observed in all treated animals. Yet, CL-MSC-GFP^* within the host myocardium (adjacent to the graft area) were found in around 62% of SASG and 50% of SASG-VATS heart sections (N & O). Quantification of the number of donor GFP⁺cells/mm² of LV scar area contiguous to the graft (n=4-5/group) showed no difference between SASG and SASG-VATS (P). Joint of the epicardium and the epicardial graft is indicated by double white arrows, while asterisks indicate the LV scar. Dotted line indicates host/graft interface. Confocal xy micrographs merged with the transmitted light channel. Scale bars, 100 pm. CL-MSC, cord-lining mesenchymal stem cells. GFP, green fluorescent protein. SD, standard deviation; and

Figure 11 : Proliferating cell nuclear antigen (PCNA) staining in the left ventricular scar area of (A) untreated rat hearts (Ml), and those treated with (B) fibrin graft through thoracotomy (FG), (C) subamnion angiogenic spheroid-enriched grafts implanted through lateral thoracotomy (SASG), (D) FG implanted by video-assisted thoracoscopic surgery (FG- VATS), arid (E) SASG delivered through VATS (SASG-VATS), 6 weeks after myocardial injury (200x). SASG and SASG-VATS-treated hearts had elevated percentage of PCNA⁺ proliferating cells in the LV scar area compared to Ml (*P<0.05, respectively). According to their morphologic appearance, PCNA⁺ cells were located predominantly in blood vessels (arrowheads) of both SASG and SASG-VATS and only in scarce cardiomyocytes of SASG- treated hearts (black arrow in C). SD, standard deviation.

Materials & Methods

Assembly of CL-MSC Angiogenic Spheroids

To produce angiogenic spheroids, 1.5x10⁴ human CL-MSC-GFP-Fluc were allowed to self- assemble for three days in 25 μΙ serum-free medium hanging drops placed in 6-well dishes' lids, and incubated at 37°C in 5% humidified C0₂. The plate's wells were filled with DPBS to avoid drop evaporation. Medium was exchanged every other day. CL-MSC spheroids were then coated at day 4 with 2x10³ HUVEC in 10 μΙ medium [10]. Angiogenic spheroids were harvested 3 days after coating (at day 7 in drop culture) and washed with sterile DPBS. Construction of Subamnion-CL-MSC Angiogenic Spheroids-enriched Grafts (SASG)

Sterile fibrin matrix (Tisseel, Baxter Healthcare Corporation, Deerfield, IL, USA) was prepared following the vendor instructions and used as scaffold material to construct SASG. For in vitro studies, 75 angiogenic spheroids (i.e. CL-MSC spheroids coated with HUVEC) in serum free media were mixed with fibrin matrix (1 :4) to a final volume of 125 μΙ, and plated into 8-well chamber slides (Lab-Tek™ll Chamber Slide™, NUNC A/S Roskilde, Denmark). This procedure was repeated upon fibrin polymerization for a final volume of 250μL· to produce 0mm X 8mm X 2.5mm grafts containing 150 spheroids distributed across the graft. Thus, 2.25x10⁶ CL-MSC and 3x10⁵ HUVEC were contained in each graft. Acellular fibrin grafts (FG) were used as negative controls. Once polymerized, grafts were covered with 0.4ml serum free media and placed in an incubator under C0₂ at 37°C. The medium was exchanged daily.

Rat Model of Myocardial Infarction

All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore and carried out in accordance with established guiding principles for animal research. Male NIH nude rats (250-300 gr, Taconic, USA) were used. Anaesthesia was induced and maintained with inhalational isoflurane (2%) and intraperitoneal injection of ketamine:xylazine (90:10mg/kg). Left-thoracotomy and pericardectomy followed by left anterior descending coronary artery (LAD) ligation was performed as previously described [15]. Carprofen (5 mg/kg, SC) and Ceftazoline (15mg/kg, bid, SC) were administered postoperatively for one week. Animals were randomly assigned a therapeutic procedure two weeks after myocardial infarction and upon confirmation of fractional area change (FAC) <40% by echocardiography. SASG Implantation via Lateral Thoracotomy

To construct grafts for in vivo studies, spheroids were mixed with DPBS and fibrin matrix to create SASG or FG with same dilution and final volume as per in vitro studies. Grafts were prepared on the same day of implantation. Following a second lateral thoracotomy as described above, SASG or FG was implanted onto the scar area using 50 μΙ of fibrin as attachment material to the epicardium. Following the therapeutic procedure the chest was closed in 3 layers, and animals were allowed to recover in a small-animal intensive care unit. Carprofen (5 mg/kg, SC) and Ceftazoline (15mg/kg, bid, SC) were administered postoperatively for 7 days.

SASG Implantation via Video-Assisted Thoracoscopic Surgery

Following general anesthesia and intubation, two 3 mm incisions were made in the rat's left hemithorax to insert the endoscopic instruments. First, a 6-0 silk suture was placed at the 4^th- 5^th intercostal space to lift the chest wall, followed by a 3mm incision made at the 4^th intercostal space, parasternal line. The second incision was made at the 8^th space, mid- axillar line. Next, a 2mm straight forward telescope (Hopkins II, 0°, Karl Storz Endoscopy, Tuttlingen, Germany) connected to a parfocal zoom camera head (TELECAM) and an integrated digital processing module (Tele Pack, all from Karl Storz) was inserted through the lower incision and advanced through the pleural space until the heart was visualized. Scar tissue between the chest wall and the left ventricular (LV) infarct area was carefully removed using a 2mm. grasping forceps (CLICKin Reddick Olsen, Karl Storz) inserted through the upper incision under video-thoracoscopic visualization.

For in situ construction of SASG via VATS, angiogenic spheroids suspended in DPBS were mixed with fibrin at the same dilution used for pre-made grafts (1 :4) and delivered onto the area of ischemia in three batches of 50 spheroids in 75μΙ_ of matrix. The mixture was loaded into a 16G catheter (Introcan Safety®, IV Catheter 16G x 2 inches, BBraun AG, Melsungen, Germany) using a 1ml syringe and was only advanced to the edge of the catheter to avoid accelerated fibrin polymerization or spheroid entrapment within the syringe. To assemble the epicardial patch in situ, each batch of fibrin-DPBS with (SASG-VATS) or without spheroids (FG-VATS) was allowed to start polymerizing within the 16G catheter for approximately 45 seconds. The catheter was then inserted into the thorax using the VATS' upper incision, and the partially gelled mixture was deposited epicardially onto the scar area under video- thoracoscopic visualization. The mixture was allowed to polymerize completely before the next batch was applied. The latter was repeated 3 times until, the scar area was totally covered with spheroids. Finally, a 100 μ!_ layer of DPBS/Fibrin was applied on top of the spheroids layer forming an in situ-created 3D graft approximately 2.5 mm thick. Following the therapeutic procedure, postoperative care was provided as described above.

Statistical Analysis

Data are presented as mean ± SD. To test for statistically significant differences between- group comparisons of echocardiographic indexes were performed using a 2-way ANOVA with repeated measures followed by pairwise comparisons by Bonferroni's post-test. The ANOVA model included control versus treatment and baseline versus 2 and 6 weeks after Ml as factors, as well as the interaction between the two factors. For other comparisons, one-way ANOVA followed by Bonferroni's-post-hoc test, and unpaired Student's t- test were used when appropriate. Differences were considered significant when P<0.05. All statistical analyses were performed using GraphPad Prism® software version 5.04 for Windows (GraphPad Software, San Diego, CA, USA).

Cell culture Umbilical cord lining-derived Mesenchymal Stem Cells (CL-MSC) isolated from the subamnion of the umbilical cord were provided by CellResearch Corporation Pte Ltd, Singapore[40]. CL-MSC were maintained in proprietary serum-free media containing DMEM-F12-CMRL1066 (GIBCO^®, Life Technologies Corporation, Carlsbad, CA, USA) with supplement of albumin, insulin, bFGF, TGFpi ( all from R&D Systems Inc, Minneapolis, MN, USA) and LIF, and incubated at 37° C in 5% humidified C0₂. CL-MSC were subcultured upon reaching confluency of 80-85% by mechanically lifting the cells from the surface of tissue culture flask using a cell lifter (Costar®, Corning Life Sciences Tewksbury, MA, USA). Human umbilical vein endothelial cells were cultured in endothelial cell medium (ScienCell Research Laboratories, Carlsbad, CA, USA). Human dermal fibroblast-adult (HDF-a) were maintained in medium 106 with low serum (GIBCO^®).

Multipotent capacity of CL-MSC

For CL-MSC adipogenic, chondrogenic and osteogenic differentiation, 6x10⁵ cells in growth medium PTT-4 were plated in 6-well plates (Techno Plastic Products AG, Trasadingen, Switzerland) and incubated at 37° C in 5% humidified C0₂. After 24 hours in culture, the appropriate differentiation medium was added (Lonza, Basel, Switzerland). Differentiation medium was changed twice a week for 3 weeks. Next, cells were stained for adipogenic (Oil Red O and hematoxylin counterstaining), chondrogenic (Alcian Blue), and osteogenic (Von ossa) differentiation as described elsewhere. [40-42] Images were acquired using an Olympus 1X71 microscope (Olympus, Japan). Production oflentiviral vectors and generation of fluorescent-bioluminescent CL-MSC

Lentiviral vectors pWPT-GFP and pLVX-LUC-puro, pseudotyped with the VSV-G, and packaged with plasmid pPax2 were generated by calcium phosphate-mediated transfection of 293T cells. Cells were plated in 10-cm plate at a density of 8X10⁶ cells in 10 ml of Dulbecco's modified Eagle's medium (DME , high-glucose, GIBCO^®) supplemented with 10% FBS, 1% Pen/Strep. Lentivirus vector, packaging, and envelope glycoprotein plasmids were mixed together with 1ml of 0.25 M CaCI2 and 1x BES (N,N-Bis(2-hydroxyethyl)-2- aminoethanesulfonic acid), incubated for 30mins and added to 293T cells. Transfection medium was replaced by complete medium 8hrs later. 24hours after the medium change, the vector-containing medium was collected and filtered through a 0.45uM pore size filter and used fresh or stored at -80°C. For transduction, 0⁵ CL-MSC cells (at either passage 3 or 4) were seeded info each well of 6-well plates. Transductions were performed in either pLVX-LUC-puro, pWPT-GFP alone, or in combination using equal volumes of viral supernatant (0.25ml and 0.5ml of each) in the presence of 8μg/ml Polybrene (Sigma, St. Louis, MO). The medium was changed to complete culture medium 12 to 16 h after addition of lentivirus. Cells transduced with pLVX-Luc-puro and pLVX-Luc-puro with WPT-GFP (CL- MSC-GFP-Fluc) were selected with O^g/ml of puromycin.

CL-MSC Immunophenotype Analysis by Flow Cytometry CL-MSC-GFP-F/uc (1 x 10⁶ cells) were harvested at passage 5-6 from culture dishes by trypsinization and washed with phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA, Invitrogen, Camarillo, CA, USA). Cells were incubated with suitable combinations of the following anti human monoclonal antibodies or isotype-matched control monoclonal antibodies (all from BD Pharmigen™, San Jose, CA, USA) unless stated otherwise: CD14-APC-Cy7 (mouse lgG2b, k clone MphiP9), CD15 (mouse IgM, k clone HI98), CD29-APC (mouse lgG1 , k clone MAR4), CD31 -Alexa Fluor® 647 ( mouse lgG2a, k clone M89D3), CD34 (mouse lgG1 clone 58, AbD Serotec, Oxford, UK), CD44 (mouse lgG1 , k clone L 78), CD45-APC (mouse lgG1 clone HI30, Molecular Probes®, Life Technologies Corporation, Carlsbad, CA, USA), CD73 (mouse lgG1 , k clone AD2), CD 90 (mouse lgG1 , k clone 5E10), CD105 (mouse lgG1 , k), CD1 17 ( mouse lgG1 clone 104D2), mouse lgG1 ,k-APC isotype control (mouse, MOPC-21), IgM, κ isotype control (mouse, clone G 155-228), lgG2a, k-Alexa Fluor® 647 isotype control (mouse, elone^"> G155-178), lgG2b, k-APC-Cy7 isotype control (mouse, clone 27-35). Cells were then fixed with 1% paraformaldehyde (PFA) and analyzed on a BD FACSCanto™ flow cytometer (BD Biosciences). Data were analyzed with FlowJo software version 10.0.4 (Tree Star, Inc, Ashland, OR, USA). Western Blot Analysis of VEGF in in CL-MSC Angiogenic Spheroids

To assess VEGF levels in angiogenic spheroids, uncoated CL-MSC spheroids were harvested after 3 days of culture in hanging drops, whereas coated spheroids were harvested either 2 days after co-culture of CL-MSC spheroids with HUVEC in hanging drops (coated spheroids day 5) or after 4 days in co-culture (coated spheroids day 7), and washed with PBS. Lysis buffer (50mM Tris-HCI at pH 7.5, 150mM NaCI, 5mM EDTA, 1% Nonidet P- 40 (NP-40) buffer and 10% glycerol) containing a protease inhibitor (Roche Diagnostics, Mannheim, Germany) was used to solubilize cells from monolayer cultures as follows: CL- MSC-GFP-Fluc, HUVEC and adult human dermal fibroblasts (HDFa, used a s negative control), or from spheroids as described above. 50 g of proteins were separated by SDS- polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto PVDF membranes (Immobilon-P, Millipore Corporation, Bedford, USA). Membranes were incubated with rabbit anti VEGF antibody (1 :500; Merck Millipore, Billerica, MA, USA) for 2 hours at room temperature, followed by incubation with rabbit anti GAPDH antibody (1 :2,500; Abeam, Cambridge, MA, USA) for 1 hour at room temperature. Membranes were then exposed using GelDoc (Bio-Rad Laboratories Inc., Hercules, CA, USA). Experiments were done in triplicate.

In vitro Evaluation of Cell viability within SASG

Because spheroids may have less access to oxygen and nutrients within SASG maintained under static culture conditions, viability of donor cells within SASG was assessed in vitro via bioluminescence imaging (BLI) after 1 , 3, and 7 days in culture. Each condition was evaluated in 3 different experiments (n=3/ time point). In vitro BLI was done using a Xenogen S Lumina System (Caliper Life Sciences, PerkinElmer Hopkinton, MA, USA) as previously described by us.[43] Bioluminescence was quantified in units of photons per second total flux (pis). Data were analyzed using Living Imaging Software version 3.2 (Caliper Life Sciences).

Detection of Apoptotic Cells within SASG

After 1 , 3 and 7 days in culture, and following in vitro bioluminescence imaging SASG were fixed in 10% buffered formalin for 2 hours, embedded in optimal cutting temperature (OCT) compound (Tissue-Tek , Sakura Finetek, Tokyo, Japan) and frozen at -80°C. To detect apoptotic cells within SASG, 10 m cryosections were stained using an active Caspase-3 antibody (Rabbit polyclonal, Abeam, Cambridge, United Kingdom) as previously described. [43] Z-stack images were obtained using a Nikon A1 R confocal microscope (Nikon, Tokyo, Japan) and subsequently processed using NIS-Elements software (v 3.1). Sections from three different experiments were analyzed. Cells were counted using Image J software (1.42q, National Institutes of Health, Bethesda, MD) and the percentage of active caspase 3-positive cells was calculated. Scanning Electron Microscopy of Subamnion-MSC Angiogenic Spheroids

For scanning electron microscopy, angiogenic spheroids were fixed with 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer (pH 7.2). Subsequently, samples were incubated in 1% osmium tetraoxide in the same buffer. Spheroids were washed and dehydrated by graded ethanol series, followed by critical-point drying with C02. Next, spheroids were mounted on aluminum stubs, and coated with a 20-nm-thick layer of gold. The samples were examined under a scanning electron microscope JEOL JSM-5600LV (JEOL Ltd, Japan).

In vivo and Ex vivo Bioluminescence Imaging

To investigate in vivo CL-MSC donor cell viability following SASG implantation, we performed BLI using a Xenogen-IVIS® Lumina imaging system (Caliper Life Sciences, PerkinElmer Hopkinton, MA, USA) as previously described.44] All rats from the SASG and SASG-VATS groups were imaged on days 1 , 3, 7 and 14, following treatment. To locate surviving CL-MSC-GFP-f/tvc 4 weeks after treatment, hearts were imaged ex-vivo immediately after animal euthanasia. Following a gentle rinse with ice-cold DPBS, explanted hearts were placed in 6-well plates, submerged in 150 mg/mL working solution of D-luciferin (Caliper Life Sciences) in DPBS and imaged for 5 minutes at 30-second intervals. Data were analyzed using Living Imaging Software version 3.2.

Subamnion-CL-MSC Angiogenic Spheroids Histology After in vitro studies, SASG were washed in PBS, fixed in 10% buffered formalin and embedded in paraffin or in optimal cutting temperature (OCT) compound (Tissue-Tek^®, Sakura Finetek, Tokyo, Japan). To identify the spheroids embedded within the graft, ten- micrometer sections were stained with Hematoxylin and Eosin (H&E) and Masson's thrichrome. Immunofluorescence of SASG

Distribution of HUVEC within the spheroids after SASG were maintained in static culture for 1 , 3 or 7 days was evaluated through immunohistochemical staining using an antibody against human CD31 (mouse monoclonal, Dako, Glostrup, Denmark). Alexa Fluor®-594 (Molecular Probes®, Life Technologies Corporation, Carlsbad, CA, USA was used as secondary antibody. A GFP-Alexa Fluor®-488 antibody (Molecular Probes®) was used to identify CL-MSC-GFP-fluc within SASG. Nuclei were counterstained with 4',6-diamidino-2- phenylindole (DAPI) (Molecular Probes®).

Echocardiography

Transthoracic echocardiography was performed by a blinded investigator (LHL) at baseline, and 2 weeks and 6 weeks following myocardial infarction (i.e. 4 weeks after treatment), using a Vivid 7 Dimension ultrasound system equipped with a broadband 10S transducer (GE VingMed, Horton, Norway). [5] LV internal diameter and wall thickness during diastole and systole were measured. End-diastolic and end-systolic cross-sectional areas were measured from the parasternal short-axis view and fractional area change (FAC) calculated as FAC% = [(end-diastolic area - end-systolic area)/ end-diastolic area] x 100, with end diastole defined as the peak of the R wave, and end systole defined as the end of the T wave. LV volumes were calculated using a modified Teichholz formula as described elsewhere. [6] Ejection fraction (EF %) was calculated as [(LV end-diastolic volume - LV end- systolic volume) /LV end-diastolic volume] x 100. Offline measurements of LV dimensions and areas were made from 3 consecutive cardiac cycles using EchoPac software (version 6, GE VingMed). Hemodynamic Measurements

Left ventricular (LV) pressure and volume measurements were performed 4 weeks post- treatment as described previously. [44] Briefly, following anesthesia, intubation and mid- thoracotomy, the ascending aorta was exposed and a 2 mm transient-time flow probe was positioned around of it for cardiac output measurement (Transonic Systems Inc, Ithaca, NY). Next, the LV was cannulated through the apex with a pressure transducer catheter (Millar Micro-Tip® model SPC-721 , Millar, Inc, TX, USA). Pressure and aortic flow waves were recorded with the Powerlab 8/30 data acquisition system (ADInstruments Pty Ltd, Castle Hill, NSW, Australia). Data were analyzed using Lab Chart Pro software (version 7.0, ADInstruments). , Left Ventricular Infarct Size and Vascularization

Infarct size was determined using Masson's Thrichrome-stained heart cross-sections from all animals which were imaged using a Nikon Eclipse TV microscope (4x objective), and a motorized stage operated with Nikon NIS-Elements AR 3.2 software. Collected images of the whole section were automatically stitched together by the NIS-Elements software. The percentage of scarred LV wall was determined using midline length measurement (calculated by dividing the midline length of the infarcted LV wall by the midline length of total LV wall)[46] using a semi-automated software (MIQuant).[47] LV regions with collagen deposition >50% of the thickness of the LV wall were considered for infarct midline calculation. [46] Epicardial tissue corresponding to the implanted graft was excluded from these measurements.

To identify proliferating cells within the LV scar area, proliferating cell nuclear antigen (PCNA) staining was done on paraffin-stained sections according to the manufacturer's instructions (PCNA staining kit, Invitrogen™, Life Technologies Corporation), and counterstained with hematoxylin. PCNA positive and negative cells were counted using Image J software (1.42q, National Institutes of Health, Bethesda, MD), followed by calculation of the percentage of PCNA⁺ cells in images taken from six random microscopic fields (100x).

After hemodynamic measurements, three animals from each group underwent direct labeling of the bloods vessels by cardiac perfusion with a lipophilic carcocyanide dye, which incorporates into endothelial cell membranes upon contact with "LV-dioctadecyl-S^S'^'- tetramethylindocarbocyanine perchlorate (Dil).[44, 48] LV vascularization was evaluated in twenty-micrometer cryosections through 0.5μηη z-stack imaging and detection of Dil+ blood vessels with a Nikon A1R confocal microscope (Nikon, Tokyo, Japan). Immunohistochemical staining of blood vessels within the LV scar area was performed in two consecutive five-micrometer cryosections (cross-sections, n=4-5 animals/group) using a ubiquitous marker for rat endothelial cells (Rat endothelial cell antigen (mouse anti-RECA-1), 1 :50 monoclonal; HyCult biotechnologt b.v, The Netherlands). Individual RECA⁺ blood vessel counts were then made on a 200 X field (20 X objective and 10X ocular; equivalent to 0.7386 mm² per 200X field). Arterioles within the LV scar area and the implanted grafts were also visualized through immunohistochemical staining in two consecutive five-micrometer heart cryosections using an antibody against smooth muscle actin (monoclonal, clone 1A4, Sigma). An antibody against a-sarcomeric actin was used to identify cardiac differentiation of donor CL-MSC (mouse monoclonal, Clone alpha-Sr-1 (Dako), whereas an antibody against human endoglin (CD105, SC-19790, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) was used to identify donor HUVEC within grafts. Alexa Fluor®-594 and Alexa Fluor®-350 (Molecular Probes®) were used as secondary antibodies and nuclei were stained with DAPI (Molecular Probes®). CL-MSC within implanted grafts or the ischemic myocardium were identified using a GFP-Alexa Fluor®-488 antibody. Quantification of the amount of donor CL-MSC-GFP⁺ (number of cells/mm²) detected within the LV scar area was done in confocal micrographs from 5 fields (100x) taken at the graft/host myocardium interface (n=5 in SASG group and n=4 in SASG-VATS group). Z-stack images from all sections were acquired at 0.5 pm intervals using a Nikon A1 R confocal microscope, and subsequently processed using NIS-Elements software (v 3.1 , Nikon).

Left ventricular blood vessel density was also quantified in all animals using Masson's trichrome- stained sections. The number of total blood vessels and arterioles (i.e. 5-50 μιτι diameter blood vessels displaying a smooth muscle media layer)[49, 50] was determined in the border zone and infarcted myocardium. Myocardium extending 0.5-1.0 mm from the infarcted tissue or infarct scar was considered to represent the border zone myocardium [51]. Micrographs from ten random high-power fields per zone (400x) were taken. All quantifications were done using Image J software.

Example 1

Phenotypic and Functional Characteristics of CL-MSC CL-MSC are plastic-adherent cells that have spindle-like, fibroblastic morphology when maintained in standard culture conditions. Multi-lineage differentiation potential of CL-MSC was confirmed, as cells were differentiable towards chondrogenic, adipogenic and osteogenic lineages (Figure 6A). Lentiviral transduction efficiency studies revealed that 75% of cells were GFP positive.

Flow cytometry studies indicated that CL-MSC-G FP-Fluc expressed the mesenchymal stem cell markers CD73 (99%), CD90 (99%), and CD105 (98%) (Figure 6B),[16] and were also positive to CD44 (38%) and CD29 (42.2%). Furthermore, CL-MSC-GFP-Fluc were negative for the endothelial cell marker CD31 (0.1%), and hematopoietic stem cell markers CD34 (0.3%), CD45 (0.3%), and C-kit/CD1 7 (0.2%). Likewise, CL-MSC-GFP-Fluc did not express the embryonic stem cell marker CD15/SSEA-1 (1.1 %). [17, 18] As previously described,¹⁶ CL-MSC-GFP-Fluc w re insignificantly positive to CD14 (6.8%) (Figure 6C). Example 2

CL'MSC spheroids coated with HUVECs display enhanced expression of VEGF dimers

By using gravity-enforced self-assembly we produced angiogenic spheroids after three days culture of cord-lining mesenchymal stem cells (CL-MSC-GFP-F/uc) followed by four additional days of coating with HUVEC. In hanging drops, cells coalesced into compact spheroids of 250-300pm diameter (Figure 7A-B). VEGF levels of CL-MSC-GFP-F/uc were comparable to HUVEC when these cells were cultured in monolayer (Supplemental Figure 7A-D). Once CL-MSC were assembled into spheroids, they expressed both VEGF monomers and dimers. This phenomenon was observed also after spheroids where coated with HUVEC for 2 and 4 days in hanging drops. We observed that VEGF monomers expression progressively reduced in spheroids and at day 7 in hanging drop culture it decreased by half of the amount seen in monolayer CL-MSC. Conversely, VEGF dimers had increased to almost double the amount seen in spheroid monomers, and remained stable throughout spheroid culture (uncoated or coated).

Example 3 Angiogenic Spheroids within SASG endure static 3D culture

Angiogenic spheroids were embedded in the fibrin matrix and distributed homogeneously within the graft (Figure 7E). SASG staining with Masson's thrichrome evidenced donor cell ECM production and deposition within the spheroids (Figure 7F). Assessment of donor cell viability within SASG via BLI following 1 , 3, and 7 days in culture revealed that CL-MSC- GFP-Fluc in our hybrid 3D graft displayed prolonged survival in spite of thick three- dimensional static culture. A non-significant decrease of CL-MSC bioluminescence was detected after 3 and 7 days of SASG culture (Figure 7G-H). In contrast, a significant increase of apoptotic cells was detected from day 1 (4.3±3.5%) to 3 (10.7±0.7%, P=0.04) and from day 1 to 7 in culture (15.8±6.3%, P=0.007) (Figure 8A-D). No difference was found in the percentage of Caspase-3⁺ cells between SASG that were 3 and 7 days in culture. Only a small amount of apoptotic cells were found within SASG after 1 day of spheroids culture within 3D grafts, which indicates that spheroids had minimal apoptosis during hanging drop culture , for a week. It is possible that some of the apoptotic cells detected within spheroids embedded in SASG corresponded to HUVEC. This could explain the slight difference with our BLI results, as photon emission was exclusively captured from CL-MSC- GFP-Fluc. Determination of cell number (DAPI⁺ cells) within angiogenic spheroids per section revealed that cell number within spheroids remained stable during SASG culture (Figure 8E). Since minimal apoptotic cells were found during the first day of SASG culture, we chose to implant SASG on the same day they were constructed in our in vivo studies. Example 4

CL-MSC within SASG organized into compact cellular networks, while HUVEC displayed angiogenic sprouting in vitro

Our in vitro studies indicated that after 1 day of spheroid culture within fibrin grafts, CL-MSC adopted a MSC in two-like elongated shape inside the spheroids and organized into compact cellular networks, while some HUVEC started to migrate towards the spheroid's core and to form sprouts (Figure 9A). At days 3 and 7 in culture HUVEC were in close contact with CL-MSC and continued progressively penetrating the spheroid's core while assembling into branches and capillary-like structures (Figure 9B-C). By day 7 of SASG static culture, CL-MSC started to migrate outside of the spheroid into the fibrin matrix. Example 5

Post-lschemic Therapy with SASG via Thoracotomy and VATS in a Rat Model

In total, ensued heart failure was confirmed 2 weeks after LAD ligation by echocardiography in 47 rats which were randomized for treatment. Except for untreated rats, grafts were delivered epicardially after 2 weeks of myocardial infarction either through thoracotomy: SASG and fibrin graft (FG) (Figure 1A); or by video-assisted thoracoscopic surgery (VATS): SASG-VATS and FG-VATS (Figure 1 B-J). The overall surgical mortality rate, defined as animal death within 4 weeks of graft implantation was 1 1.8% (four of 36 rats that underwent an implantation procedure, as follows: SASG, 0 of 8; FG, 2 of 10; SASG-VATS, 0 of 8; FG- VATS, 2 of 9). Mortality in untreated animals (from week 2 to 6 after LAD ligation) was 27.3% (3 of 11 Ml rats). Hence the number of rats that survived until retrieval (at 6 weeks after injury) was: Ml, n=8; SASG, n=8; FG, n=8; SASG-VATS, n=8 and FG-VATS, n=7.

Prospective assessment of CL-MSC-GFP-f/uc cell viability in vivo with BLI showed that donor cell survival was comparable between SASG and SASG-VATS groups (Figure 2A-C). A non-significant reduction in bioluminescence was observed during the first week after graft implantation through both techniques, followed by a significant decrease in photon emission from day 1 to 14 (8.3x10⁷±4.2x10⁷ p/s vs. 5.2x10⁵ ±1.2x10⁶ p/s, and 1.1x10⁸±1.0x10⁸ p/s vs. 2.8x10⁶±5.8x10⁶ p/s; P<0.01 and P<0.001 ). The presence of surviving CL-MSC in the LV scar area was detected 4 weeks after treatment via ex vivo BLI in explanted hearts from SASG and SASG-VATS rats (Figure 2D).

Example 6

SASG/SASG-VATS Treatment Preserved Cardiac Function and Attenuated Remodeling

Transthoracic echocardiography two weeks after induction of myocardial injury confirmed the presence of LV remodelling and deterioration of heart function in all animals, as significant increase in LV internal dimensions in systole and diastole, decrease in LV thickness and marked declines in ejection fraction (EF) and fractional shortening (FS) were found in all groups compared to baseline (Table 2). Evaluation 4 weeks after treatment (i.e. 6 weeks after myocardial infarction) revealed that therapeutic intervention with SASG led to an enhancement of wall thickness and thickening, since values in systole and diastole increased and were comparable to baseline; whereas values in systole were significantly increased compared to week 2 in the same animals (P<0.001 ). Cardiac function was preserved in treated animals, since no further deterioration in EF and FS was observed in SASG and SASG-VATS groups from week 2 to week 6 after myocardial infarction (Table 2). A trend towards enhancement in EF from week 2 to 6 after injury was observed in SASG (14.1%) and SASG-VATS (6.2%) animals, while Ml rats had a -11.4% drop in EF. FG also led to further decrease in EF by -10.53%, while FG-VATS induced a -15.77% fall in EF from week 2 to 6 after myocardial ischemia (Table 2). Comparisons between groups at end-point revealed that SASG animals displayed thicker LV walls in systole compared to Ml (P<0.001) and FG (P<0.05), while SASG-VATS animals had thicker LV walls in systole compared to Ml (PO.05), 4 weeks after treatment. Likewise, SASG and SASG-VATS hearts displayed significantly more conserved LV dimensions when compared to Ml, both in systole and diastole, at end-point (Figure 2E). Furthermore, FS was higher in SASG- and SASG-VATS- treated groups compared to Ml (P<0.0001 and P<0.01) and to their respective FG/FG-VATS control (P<0.01 and PO.05), 4 weeks after treatment. Similarly, EF in SASG animals (54.7±9.6 %) was higher than in Ml (38.9±6.9 %, PO.01 ), and FG (41.1 ±10.3%, PO.05), whereas EF in SASG-VATS animals (46.0±6.0%) was higher than Ml (PO.05), 4 weeks after treatment (Figure 2E).

SASG- and SASG-VATS-treated animals displayed significantly lower LV-end diastolic pressure compared to Ml (2.56±0.8 and 3.20±0.8 mmHg vs. 5.65±1.8 mmHg; PO.01 and P<0.05), 4 weeks after treatment (Table 1 ). Both SASG groups had better contractility (increased dP/dt max) compared their respective FG control groups and Ml rats. Interestingly, this increase was only significant in SASG-VATS (P<0.05, respectively). Cardiac output (CO) in SASG (35.6 ±10.2 mL/min), and SASG-VATS (35.1+7.9 mL/min) animals was enhanced compared to Ml rats (19.2±5.1 mL/min; P<0.001 , respectively). SASG-treated rats also had higher CO than FG-treated rats (25.1±2.8 mL/min; PO.05). Consequently, stroke volume (SV) was also significantly increased in SASG and SASG- VATS groups relative to Ml (P<0.01 and P<0.05).

Example 7

Effect of SASG and SASG-VATS Treatment on Left Ventricular Infarct Size and Vascularization

Consistent with echocardiographic evaluation, morphometric studies showed that SASG and SASG-VATS hearts had more preserved LV dimensions than the other groups (Figure 2F). Measurement of the percentage of LV containing fibrosis revealed that treatment with SASG and SASG-VATS led to smaller LV scar size (23.23±12.3% and 29.36±7.93%) relative to FG and FG-VATS controls (49.36 ±12.9% and 49.01 ±1 1.7%; P<0.01 and P<0.05), and to Ml hearts (56.21±10.2%; PO.0001 and P<0.01) (Figure 2G).

The vascular density (number of RECA-1 ⁺ blood vessels) in the LV scar area per 200x field was higher in SASG-treated hearts than FG (76.2 ± 17.9 vs. 14.0 ± 2.2, P<0.001 ) and Ml hearts (8.0 ±1.9, P<0.0001 ). Similarly, SASG-VATS hearts had more vasculature (59.6±12.4) than FG-VATS (14.0 ±2.0, P<0.01), and untreated animals (PO.01) (Figure 3A- F). Also, abundant host blood vessels (RECA-1 ⁺) were observed within the graft area of SASG- (Figure 3G) and SASG-VATS-treated hearts (Figure 3H). Likewise, a great amount of Dil⁺ functional blood vessels with seemingly active angiogenic sprouting was observed in the graft area and LV scar area of SASG and SASG-VATS hearts (Figure 3I-K). Quantification of blood vessel density on Masson's trichrome-stained sections showed similar results to our analysis using RECA-1 immunostaining, as SASG and SASG-VATS treatment led to higher blood vessel density in the LV infarct area compared to all the other groups (Figure 4A-H). This significantly higher vascularity in both groups consisted of capillaries and arterioles. SASG and SASG-VATS hearts also had increased number of arterioles at the LV border zone compared to all groups (Figure 4I). Abundant vascularization was also observed in the engrafted epicardial patch of both SASG-treated groups (Figure 4F-G and 4J-K). Consistently, a large amount of alpha-smooth muscle actin positive (a-SMA⁺) blood vessels were observed in treated groups. These arterioles were detected throughout the LV scar area and infiltrating the grafts (Figure 10A-J).

In agreement with our ex-vivo BLI data, engrafted CL-MSC-GFP⁺ were detected in both SASG and SASG-VATS hearts either within the ECM of the angiogenic spheroids or embedded in the fibrin graft. Donor CL-MSC were found within remnants of epicardial graft in all animals treated with SASG and SASG-VATS (Figure 5 and Figure 10K-O), yet the presence of CL-MSC-GFP⁺ in the host myocardium was only detected in 5 out of 8 SASG- treated animals (62 5%) and in 4 out of 8 SASG-VATS-treated rats (50%). Quantification of the number of GFP⁺ cells in the host myocardium adjacent to the graft per animal per mm² was comparable between SASG (23.2±11.4) and SASG-VATS (20.9±8.6) (P=0.78) (Figure 10P). To note, we were not able to detect donor endoglin⁺ HUVEC within the graft or the host myocardium in any of the spheroid-treated animals.

In this study, cardiomyogenic differentiation of donor CL-MSC was not detected. Yet, some of the implanted human CL-MSC contributed to vascularization, as GFP-expressing cells were colocalized with a-SMA⁺ cells implying smooth muscle differentiation of the CL-MSC delivered within angiogenic spheroids. These differentiated cells were observed in SASG and SASG-VATS groups, both within the scar area or the epicardial graft in proximity of the spheroid's ECM. However, CL-MSC cells that remained within the spheroids did not express a-SMA (Figure 5). Furthermore, SASG/SASG-VATS-treated hearts had elevated percentage of PCNA⁺ proliferating cells in the LV scar area (29.62±12.6% and 18.04±4.0%) relative to Ml (0.24±0.1%, P<0.05, respectively) (Figure 11).

Table 1 Hemodynamic parameters in infarcted untreated rats (Ml), fibrin graft- (FG), subamnion cord-lining mesenchymal stem cells angiogenic spheroids-enriched graft- (SASG), fibrin graft generated in situ via VATS- (FG-VATS), and SASG generated in situ via VATS- (SASG-VATS) treated rats, 6 weeks after myocardial injury.

Ml FG SASG FG-VATS SASG-VATS

(n= 8) (n= 8) (n= 8) (n= 7) (n= 8)

LVEDP (mmHg) 5.65+1.75 4.17±0.58 2.56+0.84"³ 4.84±2.98 3.20+0.79^*3

Mean Pressure (mmHg) 22.45±3.41 23.34±4.35 20.62±3.75 23.70±6.00 24.6614.17

Systolic Duration (s) 0.1010.01 0.10+0.02 0.10+0.01 0.09±0.01 0.0910.01

Diastolic Duration (s) 0.16+0.04 0.15±0.04 0.17±0.07 0.13±0.02 0.13+0.02

Cycle Duration (s) 0.26±0.04 0.25+0.06 0.27±0.07 0.22±0.02 0.2310.02

Heart Rate (BPM) 255.59±40.23 237.89±33.98 246.94+44.76 272.25+26.79 279.08+30.56

Max dP/dt (mmHg/s) 2867.53+618.84 2839.34+429.85 3244.06+228.33 2669.19±576.28 3781.15+867.58^*a'^*d

Min dP/dt (mmHg/s) -2170.70+415.70 -2014.07+465.05 -2247.501691.02 -2006.76+668.16 -2733.17+776.39

Tau (ms) 19.55±3.73 17.58+4.91 16.46±1.74 17.31l2.47 16.7614.68

Cardiac Output (mL/min) 19.15±5.10 25.09±2.79 35.56+10.18*³'^*" 26.30±3.77 35.07+7.94*³

Stroke volume (mL/beat) 0.08±0.03 O.lltO.02 0.14±0.04 ^a' 0.10+0.01 0.13+0.03^** ¹ VATS, video-assisted thoracoscopic surgery ;LVEDP, left ventricular end-diastolic pressure; BPM, beats per minute; s, seconds; ms, milliseconds. Statistical significance is indicated as follows: ^*a P<0.05 vs. Ml; ^"a P<0.01 vs. Ml; ^#a P<0.001 vs. Ml;^* P<0.05 vs. FG, ^*dP<0.05 vs. FG-VATS.

5

Table 2. Comparison of LV remodeling and function between infarcted untreated rats (Ml), fibrin graft- (FG), human subamnion- cord-lining mesenchymal stem cells angiogenic spheroids-enriched graft- (SASG), fibrin graft generated in situ via VATS- (FG-VATS), and SASG generated in situ via VATS- (SASG-VATS) treated rats, by two-dimensional echocardiography, before (baseline), and 2 and 6 weeks after myocardial injury.

Ml FG SASG FG-VATS SASG-VATS

(n= 8) (n= 8) (n= 8) (n= 7) (n= 8)

IVSd (cm)

Baseline 0.1510.02 0.1510.01 0.1510.01 , 0.16+0.02 0.1510.01

2 weeks post-MI 0.12+0.01 0.13+0.01 0.1210.02 0.11+0.03 0.11+0.02

P* (Baseline vs. 2 weeks) <0.01 < 0.01 <0.01 < 0.0001 < 0.0001

6 weeks post-MI 0.11±0.02 0.11+0.01 0.13+0.02 0.11+0.03 0.12+0.02

Pt (Baseline vs. 6 weeks) < 0.001 < 0.0001 NS < 0.0001 < 0.001

Pt (2 weeks vs. 6 weeks) NS NS NS NS NS

IVSs (cm)

Baseline 0.22±0.03 0.20+0.02 0.21+0.02 0.22+0.01 0.2310.01

2 weeks post-MI 0.1310.02 0.1310.02 0.13 + 0.02 0.12+ 0.05 0.1110.04

P* (Baseline vs. 2 weeks) < 0.0001 < 0. 01 < 0.0001 < 0.0001 < 0.0001

6 weeks post-MI 0.10+0.02 0.12+0.02 0.18+0.07^ea'*^b 0.11+0.05 0.13+0.04*"

Pt (Baseline vs. 6 weeks) < 0.0001 < 0. 0001 NS < 0.0001 < 0.0001

Pt (2 weeks vs. 6 weeks) NS NS <0.001 NS NS

LVIDd (cm)

Baseline 0.69+0.06 0.7010.07 0.69+0.06 0.7110.05 0.73+0.04

2 weeks post-MI 0.9010.09 0.84+0.04 0.81 +0.09 0.8410.09 0.8610.06

P* (Baseline vs. 2 weeks) < 0.0001 < 0.0001 < 0.001 < 0.001 < 0.001

6 weeks post-MI 1.01+0.07 0.92+0.03 0.88+0.08**³ 0.9110.07 0.90+0.06*"

P^† (Baseline vs. 6 weeks) < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

Pt (2 weeks vs. 6 weeks) <0.01 NS NS NS NS

LVIDs (cm)

Baseline 0.44+0.08 0.4410.07 0.43+0.05 0.42+0.04 0.44+0.04

2 weeks post-MI 0.75+0.12 0.68+0.07 0.6510.10 0.6710.13 0.74+0.05

P* (Baseline vs. 2 weeks) < 0.0001 < 0.0001 < 0.0001 < 0.0001 , < 0.0001

6 weeks post-MI 0.8510.09 0.77+0.05 0.67+0.07^l"'^a*^b 0.7510:11 0.7110.03**³

Pt (Baseline vs. 6 weeks) < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

Pt (2 weeks vs. 6 weeks) <0.05 <0.05 NS NS NS FS (%)

Baseline 37.0+8.44 37.0 +5.12 38.2±3.72 40.1+2.72 41.3±4.74

2 weeks post-MI 17.7 ± 4.77 19.1 + 6.96 20.0±6.17 21.1+8.25 18.5±3.21

P* (Baseline vs. 2 weeks) < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

6 weeks post-MI 15.6+.2.96 16.814.84 24.5i3.46** ³' **^b 17.1+5.49 23.214.44* *^a' *^d

Pt (Baseline vs. 6 weeks) < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

Pt (2 weeks vs. 6 weeks) NS NS NS NS NS

EF (%)

Baseline 77.114.82 74.616.25 76.214.26 77.8+3.32 78.714.36

2 weeks post-MI 43.8+10.2 , 46.0+13.0 48.0111.8 47.8+12.1 43.315.61

P* (Baseline vs. 2 weeks) < 0.0001 < 0.001 < 0.0001 < 0.0001 < 0.0001

6 weeks post-MI 38.916.85 41.1110.3 54.7+9.6^Sa'*^b 40.3+11.7 46.0+5.97*³

Pt (Baseline vs. 6 weeks) < 0.0001 < 0.0001 < 0.0001 <0.0001 < 0.0001

Pt (2 weeks vs. 6 weeks) NS NS NS NS NS

Ml F6 SASG FG-VATS SASG-VATS (n= 8) (n= 8) (n= 8) (n= 7) (n= 8)

LVAd (cm²)

Baseline 0.45+0.15 0.4210.11 0.3910.05 0.5010.05 0.50+0.05

2 weeks post-MI 0.691 0.14 0.61+ 0.08 0.62 + 0.11 0.68 1 0.13 0.68 10.10

P* (Baseline vs. 2 weeks) < 0.0001 < 0.001 < 0.001 < 0. 01 < 0. 001

6 weeks post-MI 0.8110.18 0.6810.07 0.6510.12 0.7510.14 0.6910.12

Pt (Baseline vs. 6 weeks) < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.001

Pt (2 weeks vs.6 weeks) NS NS NS <0.01 NS

LVAs (cm^z)

Baseline 0.16+0.07 0.1910.06 0.16+0.04 0.21 +0.03 0.19 10.05

2 weeks post-MI 0.41 +0.13 0.38 10.04 0.39 +0.09 0.4410.11 0.45 +0.09

P* (Baseline vs. 2 weeks) < 0.0001 < 0.001 < 0.0001 < 0. 01 < 0. 001

6 weeks post-MI 0.54 +0.18 0.45 10.07 0.39 +0.09*^a 0.49 +0.12 0.46 +0.08

Pt (Baseline vs. 6 weeks) < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.001

Pt (2 weeks vs. 6 weeks) <0.01 NS NS NS NS

FAC (%)

Baseline 59.0+5.13 58.6+4.84 59.715.96 57.2+4.75 61.0+9.35

2 weeks post- I 33.1+3.84 36.514.72 37.113.28 35.7+5.38 33.614.34

P* (Baseline vs. 2 weeks) < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

6 weeks post-MI 28.915.79 31.1+7.14 41.6+4.74"^a' *^b 34.5+5.49 35.812.22

Pt (Baseline vs. 6 weeks) < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

Pt (2 weeks vs. 6 weeks) NS NS NS NS NS

VATS, video-assisted thoracoscopic surgery; LV, left ventricular; Ml; myocardial infarction; IVSd, LV wall thickness/ interventricular septum dimensions in diastole; IVSs, LV wall thickness/ interventricular septum dimensions in systole; LVIDd, LV end internal dimension in diastole; LVIDs, LV end internal dimension in systole; FS, LV fractional shortening; EF, LV ejection fraction; LVAd, LV area in diastole;

LVAs, LV area in systole, FAC, LV fractional area change. Values are presented as mean ± SD; P^*, p- values derived from paired comparisons between baseline and 2 weeks after Ml measurements; P†, p-values derived from paired comparisons between baseline and 6 week after Ml (4 weeks after treatment) measurements; Ρφ p-values derived from paired comparisons between 2 weeks (pre- treatment) and 6 weeks after Ml (4 weeks post-treatment) measurements. Group-time interaction: *a P<0.05 vs. Ml; **a P<0.01 vs. Ml; #a P<0.001 vs Ml; ##a P<0.0001 vs. Ml; *b P<0.05 vs. FG; **b P<0.005 vs. FG; *d P<0.05 vs. FG-VATS

Strauer BE and G Steinhoff. (2011). 10 years of intracoronary and intramyocardial bone marrow stem cell therapy of the heart: from the methodological origin to clinical practice. J Am Coll Cardiol 58:1095-104.

Iso Y, JL Spees, C Serrano, B Bakondi, R Pochampally, YH Song, BE Sobel, P Delafontaine and DJ Prockop. (2007). Multipotent human stromal cells improve cardiac function after myocardial infarction in mice without long-term engraftment. Biochem Biophys Res Commun 354:700-6.

Bartosh TJ, Z Wang, AA Rosales, SD Dimitrijevich and RS Roque. (2008). 3D-model of adult cardiac stem cells promotes cardiac differentiation and resistance to oxidative stress. J Cell Biochem 105:612-23.

Lee EJ, SJ Park, SK Kang, GH Kim, HJ Kang, SW Lee, HB Jeon and HS Kim. (2012). Spherical bullet formation via E-cadherin promotes therapeutic potency of mesenchymal stem cells derived from human umbilical cord blood for myocardial infarction. Mol Ther 20:1424-33.

Korff T and HG Augustin. (1998). Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation. J Cell Biol 143:1341-52. Banfi A, A uraglia, B Dozin, M Mastrogiacomo, R Cancedda and R Quarto. (2000). Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy. Exp Hematol 28:707- 15.

Frith JE, B Thomson and PG Genever. (2010). Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential. Tissue Eng Part C Methods 16:735-49.

Scadden DT. (2006). The stem-cell niche as an entity of action. Nature 441:1075-9. Anderer U and J Libera. (2002). In vitro engineering of human autogenous cartilage. J Bone Miner Res 17:1420-9.

Kelm JM, V Djonov, SP Hoerstrup, CI Guenter, LM Ittner, F Greve, A Hierlemann, CD Sanchez-Bustamante, JC Perriard, E Ehler and M Fussenegger. (2006). Tissue- transplant fusion and vascularization of myocardial microtissues and macrotissues implanted into chicken embryos and rats. Tissue Eng 12:2541-53.

Kelm JM, C Diaz Sanchez-Bustamante, E Ehler, SP Hoerstrup, V Djonov, L Ittner and M Fussenegger. (2005). VEGF profiling and angiogenesis in human microtissues^ J Biotechnol 118:213-29.

Deuse T, M Stubbendorff, K Tang-Quan, N Phillips, MA Kay, T Eiermann, TT Phan, HD Volk, H Reichenspurner, RC Robbins and S Schrepfer. (201 1). Immunogenicity and immunomodulatory properties of umbilical cord lining mesenchymal stem cells. Cell Transplant 20:655-67.

Bueno H RJSWY and et al. (2010). Trends in length of stay and short-term outcomes among medicare patients hospitalized for heart failure, 1993-2006. JAMA 303:2141- 2147.

Lilyanna S, EC Martinez, TD Vu, LH Ling, SU Gan, AL Tan, TT Phan and T Kofidis. (2013). Cord lining-mesenchymal stem cells graft supplemented with an omental flap induces myocardial revascularization and ameliorates cardiac dysfunction in a rat model of chronic ischemic heart failure. Tissue Eng Part A 19:1303-15.

Martinez EC, J Wang, S Lilyanna, LH Ling, SU Gan, R Singh, CN Lee and T Kofidis. (2013). Post-ischaemic angiogenic therapy using in vivo prevascularized ascorbic acid-enriched myocardial artificial grafts improves heart function in a rat model. J Tissue Eng Regen Med 7:203-12.

Dominici M, K Le Blanc, I Mueller, I Slaper-Cortenbach, F Marini, D Krause, R Deans, A Keating, D Prockop and E Horwitz. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315-7.

Kofidis T, JL de Bruin, G Hoyt, DR Lebl, M Tanaka, T Yamane, CP Chang and RC Robbins. (2004). Injectable bioartificial myocardial tissue for large-scale intramural cell transfer and functional recovery of injured heart muscle. J Thorac Cardiovasc Surg 128.571-8.

Vu DT, LK Ti, LC Ong, PH Neo, CN Lee and T Kofidis. (2012). Novel sutureless mitral valve implantation method involving a bayonet insertion and release mechanism: A proof of concept study in pigs. J Thorac Cardiovasc Surg 143:985-8. Schuleri KH, GS Feigenbaum, M Centola, ES Weiss, JM Zimmet, J Turney, J Kellner, MM Zviman, KE Hatzistergos, B Detrick, JV Conte, I McNiece, C Steenbergen, AC Lardo and JM Hare. (2009). Autologous mesenchymal stem cells produce reverse remodelling in chronic ischaemic cardiomyopathy. Eur Heart J 30:2722-32.

20. Williams AR, B Trachtenberg, DL Velazquez, I McNiece, P Altman, D Rouy, AM Mendizabal, PM Pattany, GA Lopera, J Fishman, J,P Zambrano, AW Heldman and

JM Hare. (201 1). Intramyocardial stem cell injection in patients with ischemic cardiomyopathy: functional recovery and reverse remodeling. Circ Res 108:792-6.

21. Wang W, Q Jiang, H Zhang, P Jin, X Yuan, Y Wei and S Hu. (201 1). Intravenous administration of bone marrow mesenchymal stromal cells is safe for the lung in a chronic myocardial infarction model. Regen Med 6:179-90.

22. Li SH, TY Lai, Z Sun, M Han, E Moriyama, B Wilson, S Fazel, RD Weisel, T Yau, JC Wu and RK Li. (2009). Tracking cardiac engraftment and distribution of implanted bone marrow cells: Comparing intra-aortic, intravenous, and intramyocardial delivery. J Thorac Cardiovasc Surg 137:1225-33 el

23. Tomita S, DA Mickle, RD Weisel, ZQ Jia, LC Tumiati, Y Allidina, P Liu and RK Li.

(2002). Improved heart function with myogenesis and angiogenesis after autologous porcine bone marrow stromal cell transplantation. J Thorac Cardiovasc Surg 123:1 132-40.

24. Kinnaird T, E Stabile, MS Burnett, CW Lee, S Barr, S Fuchs and SE Epstein. (2004).

Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res 94:678-85.

25. Reffelmann T, M Dorr, H Volzke, J Kors, J Ruppert, D Robinson and SB Felix.

(2009). Combination of electrocardiographic and echocardiography information identifies individuals prone to a progressive increase in left ventricular mass over 5 years. J Hypertens 27:861-8.

26. Huang NF, A Lam, Q Fang, RE Sievers, S Li and RJ Lee. (2009). Bone marrow- derived mesenchymal stem cells in fibrin augment angiogenesis in the chronically infarcted myocardium. Regen Med 4:527-38.

27. Khan M, S Akhtar, S Mohsin, NK S and S Riazuddin. (201 1 ). Growth factor preconditioning increases the function of diabetes-impaired mesenchymal stem cells. Stem Cells Dev 20:67-75.

28. Khan M, S Mohsin, SN Khan and S Riazuddin. (201 1). Repair of senescent myocardium by mesenchymal stem cells is dependent on the age of donor mice. J Cell Mol Med 15:1515-27.

29. Hare JM, JE Fishman, G Gerstenblith, DL DiFede Velazquez, JP Zambrano, VY Suncion, M Tracy, E Ghersin, PV Johnston, JA Brinker, E Breton, J Davis-Sproul, IH Schulman, J Byrnes, AM Mendizabal, MH Lowery, D Rouy, P Altman, C Wong Po Foo, P Ruiz, A Amador, J Da Silva, IK McNiece and AW Heldman. (2012). Comparison of allogeneic vs autologous bone marrow-derived mesenchymal stem cells delivered by transehdocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial. JAMA 308:2369-79.

Kelm JM, M Breitbach, G Fischer, B Odermatt, I Agarkova, BK Fleischmann and SP Hoerstrup. (2012). 3D microtissue formation of undifferentiated bone marrow mesenchymal stem cells leads to elevated apoptosis. Tissue Eng Part A 18:692-702. Lee WY, HJ Wei, JJ Wang, KJ Lin, WW Lin, DY Chen, CC Huang, TY Lee, HY Ma, SM Hwang, Y Chang and HW Sung. (2012). Vascularization and restoration of heart function in rat myocardial infarction using transplantation of human cbMSC/HUVEC core-shell bodies. Biomaterials 33:2127-36.

Reffelmann T, S Konemann and RA Kloner. (2009). Promise of blood- and bone marrow-derived stem cell transplantation for functional cardiac repair: putting it in perspective with existing therapy. J Am Coll Cardiol 53:305-8.

van der Spoel Tl, SJ Jansen of Lorkeers, P Agostoni, E van Belle, M Gyongyosi, JP Sluijter, MJ Cramer, PA Doevendans and SA Chamuleau. (2011). Human relevance of pre-clinical studies in stem cell therapy: systematic review and meta-analysis of large animal models of ischaemic heart disease. Cardiovasc Res 91 :649-58.

Siegel G, P Krause, S Wohrle, P Nowak, M Ayturan, T Kluba, BR Brehm, B Neumeister, D Kohler, P Rosenberger, L Just, H Northoff and R Schafer. (2012). Bone marrow-derived human mesenchymal stem cells express cardiomyogenic proteins but do not exhibit functional cardiomyogenic differentiation potential. Stem Cells Dev 21 :2457-70.

Quevedo HC, KE Hatzistergos, BN Oskouei, GS Feigenbaum, JE Rodriguez, D Valdes, PM Pattany, JP Zambrano, Q Hu, I McNiece, AW Heldman and JM Hare. (2009). Allogeneic mesenchymal stem cells restore cardiac function in chronic ischemic cardiomyopathy via trilineage differentiating capacity. Proc Natl Acad Sci U S A 106:14022-7.

da Silva Meirelles L, Al Caplan and NB Nardi. (2008). In search of the in vivo identity of mesenchymal stem cells. Stem Cells 26:2287-99.

Potapova IA, PR Brink, IS Cohen and SV Doronin. (2008). Culturing of Human Mesenchymal Stem Cells as Three-dimensional Aggregates Induces Functional Expression of CXCR4 That Regulates Adhesion to Endothelial Cells. J Biol Chem 283:13100-13107.

Bartosh TJ, JH Ylostalo, A Mohammadipoor, N Bazhanov, K Coble, K Claypool, RH Lee, H Choi and DJ Prockop. (2010). Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci U S A 107:13724-9.

39. Takagawa J, Y Zhang, ML Wong, RE Sievers, NK Kapasi, Y Wang, Y Yeghiazarians, RJ Lee, W Grossman and ML Springer. (2007). Myocardial infarct size measurement in the mouse chronic infarction model: comparison of area- and length-based approaches. J Appl Physiol 102:2104-11.

40. Cheong HH, J Masilamani, TT Phan and SY Chan. (2010). Cord lining progenitor cells: potential in vitro adipogenesis model. Int J Obes 34:1625-1633.

41. Kofidis T, JL de Bruin, G Hoyt, DR Lebl, M Tanaka, T Yamane, CP Chang and RC Robbins. (2004). Injectable bioartificial myocardial tissue for large-scale intramural cell transfer and functional recovery of injured heart muscle. The Journal of thoracic and cardiovascular surgery 128:571-8.

42. Deuse T, M Stubbendorff, K Tang-Quan, N Phillips, MA Kay, T Eiermann, TT Phan, HD Volk, H Reichenspurner, RC Robbins and S Schrepfer. (20 ). Immunogenicity and immunomodulatory properties of umbilical cord lining mesenchymal stem cells. Cell transplantation 20:655-67.

43. Martinez EC, J Wang, SU Gan, R Singh, CN Lee and T Kofidis. (2010). Ascorbic acid improves embryonic cardiomyoblast cell survival and promotes vascularization in potential myocardial grafts in vivo. Tissue engineering. Part A 16:1349-61. 44. Martinez EC, J Wang, S Lilyanna, LH Ling, SU Gan, R Singh, CN Lee and T Kofidis. (2011 ). Post-ischaemic angiogenic therapy using in vivo prevascularized ascorbic acid- enriched myocardial artificial grafts improves heart function in a rat model. Journal of tissue engineering and regenerative medicine.

45. Weytjens C, B Cosyns, J D'Hooge, C Gallez, S Droogmans, T Lahoute, P Franken and G Van Camp. (2006). Doppler myocardial imaging in adu|t male rats: reference values and reproducibility of velocity and deformation parameters. Eur J Echocardiogr 7:411-7.

46. Takagawa J, Y Zhang, ML Wong, RE Sievers, NK Kapasi, Y Wang, Y Yeghiazarians, RJ Lee, W Grossman and ML Springer. (2007). Myocardial infarct size measurement in the mouse chronic infarction model: comparison of area- and length-based approaches. Journal of applied physiology 102:2104-11.

47. Nascimento DS, M Valente, T Esteves, F de Pina Mde, JG Guedes, A Freire, P Quelhas and OP Pinto-do. (2011). MIQuant-semi-automation of infarct size assessment in models of cardiac ischemic injury. PloS one 6:e25045. 48. Li Y, Y Song, L Zhao, G Gaidosh, AM Laties and R Wen. (2008). Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye Dil. Nat Protoc 3:1703-8.

49. Al-Khaldi A, N Eliopoulos, D Martineau, L Lejeune, Lachapelle and J Galipeau. (2003). Postnatal bone marrow stromal cells elicit a potent VEGF-dependent neoangiogenic response in vivo. Gene Ther 10:621-9. . ^·

50. Crottogini A, PC Meckert, G Vera Janavel, E Lascano, J Negroni, H Del Valle, E Dulbecco, P Werba, L Cuniberti, V Martinez, A De Lorenzi, J Telayna, A Mele, JL Fernandez, L Marangunich, M Criscuolo, MC Capogrossi and R Laguens. (2003). Arteriogenesis induced by intramyocardial vascular endothelial growth factor 165 gene transfer in chronically ischemic pigs. Hum Gene Ther 14:1307-18.

51. Jin P, E Wang, YH Wang, W Huang, W Kuang, C Sun, S Hu and H Zhang. (2012). Central zone of myocardial infarction: a neglected target area for heart cell therapy. J Cell Mol Med 16:637-48.

52. Kelm JM, C Diaz Sanchez-Bustamante, E Ehler, SP Hoerstrup, V Djonov, L Ittner and M Fussenegger. (2005). VEGF profiling and angiogenesis in human microtissues.

Journal of biotechnology 1 18:213-29.

Claims

1 A therapeutic cell composition comprising: a spheroid comprising lineage restricted stem cells derived from pluripotent stem cells or multipotent stem cells and activated endothelial cells wherein the combined cell preparation is associated with a biodegradable cell support matrix.

2. The composition according to claim 1 wherein said lineage restricted stem cells are derived from induced pluripotent stem cells.

3. The composition according to claim 1 wherein said lineage restricted stem cells are derived from embryonic stem cells 4. The composition according to claim 3 wherein said lineage restricted embryonic stem cells are derived from human embryonic stem cells.

5. The composition according to claim 1 wherein said multipotent stem cells are cardiac stem or progenitor cells.

6. The composition according to claim 1 wherein said multipotent stem cells are mesenchymal stem cells.

7. The composition according to claim 6 wherein said mesenchymal stem cells are selected from the group consisting of: bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord blood mesenchymal stem cells or sub-amniotic cord-lining mesenchymal stem cells. 8. The composition according to claim 7 wherein said stem cells ar,e angiogenic stem cells.

9. The composition according to claim 8 wherein said angiogenic stem cells are mesenchymal stem cells

10. The composition according to claim 9 wherein said angiogenic mesenchymal stem cells are sub-amniotic cord-lining mesenchymal stem cells.

11. The composition according to any one of claims 1 to 10 wherein said activated endothelial cells are umbilical vein endothelial cells. 2. The composition according to any one of claims 1 to 10 wherein said mesenchymal stem cells are mouse, rat, primate or human.

13. The composition according to claim 12 wherein said mesenchymal^* stem cells are human.

14. The composition according to any one of claims 1-13 wherein said cell support matrix polymerises at body temperature. 15. The composition according to claim 14 wherein the cell support matrix comprises natural or synthetic polymers.

16. The composition according to claim 15 or 16 wherein said cell support matrix comprises a hydrogel.

17. The composition according to any one of claims 1- 13 wherein said cell support matrix comprises or consists essentially of fibrin. 8. A composition according to any one of claims 1-17 for use in the treatment of heart disease.

19. The composition according to claim 18 wherein heart disease is selected from the group: ischemic heart disease, coronary heart disease, congestive heart failure, cardiomyopathy or myocardial infarction.

20. The composition according to claim 19 wherein said heart disease is ischemic heart disease.

21. A surgical procedure to repair heart tissue in a subject in need of heart surgery comprising the steps: i) providing a composition according to any one of claims 1-17;

ii) preparing an incision in the thoracic region of the subject thereby exposing the heart;

iii) contacting the heart tissue in need of repair with the composition of (i) above; and optionally

iv) repeat step (iii).

22. The method according to claim 21 wherein said surgical procedure is video-assisted thoracoscope surgery.

23. The method according to claim 21 or 22 wherein the composition is applied to the heart tissue via a catheter wherein the composition is not fully polymerized. 24. The method according to any one of claims 21-23 wherein said subject is human.

25. The method according to any one of claims 21-24 wherein the repaired heart tissue is a result of a heart disease selected from the group: ischemic heart disease, coronary heart disease, congestive heart failure, cardiomyopathy or myocardial infarction.

26. The method according to claim 25 wherein said heart disease is ischemic heart disease.

27. A method for the preparation of angiogenic spheroids comprising lineage restricted stem cells derived from pluripotent stem cells or multipotent stem cells comprising the steps: i) providing a growing culture of stem cells in cell culture medium;

iii) contacting the induced angiogenic stem cells with a preparation comprising activated endothelial cells to form a combined cell preparation.

28. The method according to claim 27 wherein said lineage restricted stem cells are derived from induced pluripotent stem cells. 29. The method according to claim 27 wherein said lineage restricted stem cells are derived from embryonic stem cells.

30. The method according to claim 29 wherein said embryonic stem cells are human embryonic stem cells.

31. The method according to claim 27 wherein said multipotent stem cells are cardiac stem or progenitor cells.

32. The method according to claim 27 wherein said multipotent stem cells are mesenchymal stem cells;

33. The method according to any one of claims 27-31 wherein said stem cells are angiogenic stem cells. 34. The method according to claim 32 wherein said mesenchymal stem cells are sub- amniotic cord-lining mesenchymal stem cells.

35. The method according to any one of claims 27 to 32 wherein said combined preparation is associated with a biodegradable cell support matrix.

36. The method according to claim 35 wherein said cell support matrix polymerises at body temperature. The method according to claim 36 wherein the cell support matrix comprises natural or synthetic polymers.

38. The method according to claim 36 or 37 wherein said cell support matrix comprises a hydrogel. 39. The method according to claim 35 wherein said cell support matrix comprises or consists essentially of fibrin.

40. The method according to any one of claims 21 to 39 wherein said hypoxic conditions to induce expression of pro-angiogenic factors is a hanging drop cell culture.

41. The method according to any one of claims 21 to 40 wherein angiogenic spheroids are harvested when their diameters reach about 250-300pm.

42. The method according to any one of claims 21 to 41 wherein angiogenic spheroids are cultured for up to about 3 days prior to combining with activated endothelial cells.

43. The method according to claim 42 wherein the combined cell preparation is cultured for up to about 4 days. 44. A kit comprising:

") lineage restricted stem cells;

■i) activated endothelial cells;

iii) a biodegradable cell support matrix; and optionally

iv) cell growth medium for the maintenance of said stem cells and/or activated endothelial cells.

45. The kit according to claim 44 wherein said lineage restricted stem cells are derived from the group consisting of: pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, multipotent stem cells, cardiac stem cells, cardiac progenitor cells or mesenchymal stem cells. 46. The kit according to claim 45 wherein said mesenchymal stem cells are selected from the group: bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord blood mesenchymal stem cells or sub-amniotic cord-lining mesenchymal stem cells

47. The kit according to claim 46 wherein the mesenchymal stem cells are sub-amniotic cord-lining mesenchymal stem cells.

48. The kit according to any one of claims 44-47 wherein said kit further comprise products used in the surgical procedure according to the invention.