GB2389589A - Pre-conditioning of heart tissue - Google Patents

Abstract

A method for pre-conditioning heart cells comprising growing said cells on an acellular silicon rubber matrix and subjecting to cycles of computer controlled stretch and relaxation. The cells are supplied with growth factors and integrin cell receptors.

Description

A biotissue engineering technique for preparing human cells for transplant

into damaged human hearts, which includes an extracellular scaffold and a culture medium for cell growth.

Backeround The human heart is composed of highly differentiated cardiac myocytes which constitute parenchyma and stroma or connective tissue. Both phylogenetically and ontogenetical]y, the marurnalian myocardium is a developmental derivative of mesoderrnal epithelium. Differentiated cardiomyocytes and epithelial cells express biochemically and immunochemically, similar types of proteins in intercellular contacts (Franke W.W. et al. 1982). More precisely, desmoplakirs of epithelial and myocardial desmosomes are immunologically and biochemically related.

Desmosomes between the cells are formed early in heart development before formation of myofibrils. Dunng development, mononucleated myoblasts fuse to form linear aggregates which themselves fuse to form parallel myotubes, later maturing into an unbranched myofibers.

The cardiomyocytes are organized into layers of rod-shaped cells parallel to each other and connected by gap junctions and intercalated discs, critical components for efficient pumping by the heart.

Repetitive stretch/relaxation cycles occur as the heart pumps blood. Hence, cardiomyocytes are subjected to continuous mechanical loading throughout their life, which since the cells are post mitotic soon after brth, is the Iife span of the individual.

The parenchyrnal cells of the heart are characterized by highly specialized finctions and loss of reproductive capacity. The acquisition of specialized functions and entailed loss of prolferative competence are collectively termed 'terminal differentiation'. The irreversible growth arrest of terminally differentiated (post-mitotic) cells is qualitatively different from reversible quiescence. Post mitotic cells do not respond with proliferation to growth factors even though they possess receptors for them.

Fibroblasts are the principal collagen-producing cells in the heart, producing the major ilbrillar collagens (Type I and Type m) same as skinType II[ in the papillary dermis, Type I in the reticular dennis). Cells adhere to collagen matnces through ECM receptors-the integrins. Specific ntegrin dimers bnd to collagen. Binding of integrins to the ECM stimulates ntracellular signaling through integrin clustering and formation of focal adhesion sites. Thus collagen acts as a receptor ligand to stimulate cellular responses e.g. cell growth, motilty and differentiation. Because these integrins are the direct contact between cells and the ECM, they might be important mechanotransducers, and in turn growth factors can modify the cell phenotype by altering fibronectin- integrin interaction (Fujio et al.l993).

The extracellular matrix synthesized by the interstitial fibroblasts probably plays an important role in the organization of the cardiomyocytes in response to stretch

(similarlyin skeletal muscle). Carver et al. (1991) Circ. Res.69, 116-122, have reported that mechanical stimulation of cardiac fibroblasts In vitro stimulate specific exracellular max components which are involved in cell attachment and organogenesis. It has been reported that mechanical strain induces growth of vascular smooth muscle cells via an autocrine action of platelet derived growth factor (PDGF) (Wilson et al. 1993). Furthermore, Sadoshima and lzumo (1993) have established that mechanical stretch activated multiple signal transduction pathways in cardiomyocytes.

Consider the table below: _ Human endothelial cells Extracellular Secretions 1. Aortic, adult vena cave Type IV procollagen, thrombospondin and fibronectin.

2. Umbilical vein Type IV procollagen, thrombospondin and fibronectin.

3. Neonatal/Adult dermis Type IV procollagen, thrombospondin, fibronectin and larninin 4. $*Neonatal/adult skin **Larnimn, type IV collagen, perlecan, nidogen/entactln fibroblasts _ and keratinocyte growth factor (KGF).

5. *Epidermal *Type procollagen, type IV & VII collagen, larninin, keratnocytes fibronectin and vascular endothelial growth factor/ vascular permeability factor (VEGF/VPF).

References: 1. & 2. Sage and Bornstein 1982; Fry et ah 1984 3. Kramer et al. 1985 4. Woodley et al. 1988, Fleischmajer et al. 1998 5. O'Keefe et al. 1984; Petersen et al. 1988; Steen and Malhotra 1992; Ballaun et al. 1995 The matrix underlying vascular endothelial cells (ECM) is similar in organization and macromolecular composition to basement membranes. The macromolecular composition of endothelial cell matrices (ECMs) reportedly include collagen types I, m, IV, v, VI, fibronectin, laminin, heparan sulfate and dermatan sulfate/chondroitn sulfate proteog)ycans, entactin, elastin with sequestered basic fibroblast growth factor (b-FGF), plasminogen activator and heparanase activity (Gospodarowicz et al. 1980a; Kosher et al. 1993).

Human umbilical vein endothelial cells maintained on the ECM need b-FGE to become confluent (Gospodarowicz et al. 1 980b), and HUVECs seeded on HUVECs ECM also need an endothelial cell growth supplement to reach confluence (Solomon 1992).

Trypsinized epiderrnal adult leratinocytes attached to HllVECs ECM in 20 minutes and n _ _:__ _ _ _, _ À. À.

mesenchyrnal interaction previously thought to be tissue-specific (Solomon 2002). The arrival of keratinocyte-produced laminin at confluence was thought to be the trigger of the matrix remodeling (by triggering the release of a sequestered ECM component or activating an integrin signaling mechanism) since HWECs do not secrete laminin (see table above). Colonies of epiderrnal keratinocytes from Dispase digested human epidermis grew amidst dermal microvascular endothelial cells and dermal fibroblasts.

Hwnan aortic smooth muscle cells (HASMCs) do attach and proliferate on HUVECs ECM - unpublishedolomon 1994 Indeed, HASMCs were used as negative controls in endothelial cell specific identity tests.

Derrnal fibroblasts secrete the same fibrillar cardiac collagens and basic fibroblast growth factor (b-FGF) present in adult heart myocyte basement membrane.

Interestingly, the alignment of myoblasts on ultrafine gratings coated with poly-L-lysine overlaid with laminin facilitated end-to end fusion and oriented myotube formation with the inhibition of lateral fusion (Clark et al. 2002). These authors seemed unaware of H. H. Vandenburgh's many publications, particularly his 1995 chapter entitled 'Response of neonatal rat cardiomyocytes to repetitive mechanical stimulation in vitro' ----see References. He was studying cardiomyocyte organogenesis and mechanogenc transduction processes involved in the neonatal to adult physiological grown process. He demonstrated that the rat neonatal cardiomyocytes could orgaruze in parallel arrays of rod shaped cells, if stretched in one direction, or perpendicular to the direction of the substratum stretch, if stretched and relaxed in a computer assisted vertical mechanical cell stimulator.

S( Invention Silicone rubber membranes will be coated with gelatin. The cells will be grown in the wells (circular) of a computerized vertical mechanical cell stimulator* (Cell Kinetics, Inc., Providence, Rl). The stainless steel cell growth chamber contains 36 Teflon-lined culture wells I Srnrn in diameter. The cell stimulator has a built-in computercontrolled stepper motor which moves prongs up and down to stretch and relax the cells in 30-35 Am increments. The cells in the mechanical cell stimulator are incubated at 37 C in a 5% CO2 incubator (see H.H. Vandenburgh et al. (1995) Ann. N.Y. Acad. Sci. 752, 19-29).

Human umbilical vein endothelial cells (HUVECs) from a single prescreened umbilical cord will seeded on top of the gelatin-coated rubber membrane and fed every 2-3 days with culture medium (Medium 199 + soluble growth factor supplement + penicillin/streptomycin) and incubated at 37 C. Conditioned medium will be saved (HUVECs CM).

At post confluence (2 days after normal confluence), the cell layer will be treated with 5 mM EDTAIDPBS ( ethylene diarnine tetra-acetic acid/Dulbecco's phophate buffered saline) at pH 7.4 until the substratum is acellular. The sub-endothelial extracellular matrix (ECM) will be left behind in an intact state (HUVECs ECM)-see Solomon D.E. (1992) in References.

Segments of pre-screened derrnal skin tissue will be severely abraded with a surgical scalpel and debris shaken offthe blade into culture medium. This solution will be layered over the HUVECs ECM and the new growth of dermal fibroblasts will be fed with conditioned medium obtained from the growth of the HUVECs above (HWECs Chit).

In turn, conditioned medium from the dermal fibroblast cell layer will be saved (DF-CM).

After a suitable period, the fibroblast cell layer will be retracted with 5 mM EDTA/DPBS (pH 7.4).

On this acellular matnx, (underlaid by the HUVECs ECM), myoblasts (or other cell types) will be seeded.

A mixture of conditioned media obtained from the dermal fibroblast cell layer (DF-CM) and the HUVECs CM will be the culture medium, when mixed according to the (v/v) ratio of 80% (HUVECs CM): 20% (DF-CM).

Tranplanted cells (e.g. myoblasts) after a period of growth will be subjected to a computer-generated pattern of stretch-relaxation cycles to simulate the beating heart. This process will pre-condition the cells prior to transplanting them via catheter technology.

Release of soluble creatine kinase, (a marker of myocardial damage) will be used as a measure o f stretch-induced damage in vitro, while the release of particulate creatine kinase into the culture medium will be a measure of the extent of cell detachment Mom the stretching substratum. This evaluation will be done if pre-screened human fete] or adult cardiomyocytes are used.

À *A Flexercel stress unit-a computer controlled vacuum unit (15-20 kPA) À may also be used with Flex culture plates (Flexcell Corp., McKeesport, À PA.)-see Wilson et al (1993) in References.

SUMMARY

A method is described for pre-conditioning human cells before transplanting them into damaged hearts. Even if stem cells were induced to form cardiomyocytes, the contention is that they would Deed to be subjected to cycles of stretch and relaxation before being used. The heart is a continuous pumping organ. By pre-conditioniDg cells, it is hoped that tissue remodeling would be facilitated by supplying them on a extracellular matrix scaffold with sequestered growth factors and localized integrin cell receptors.

Claims (3)

CLAIMS It is envisaged that transplanting the cells on an acellular extracellular matrix scaffold after preconditioning them with stretch/relaxation cycles will lead to cardiac tissue remodellin and subsequently, repair of the damaged heart. Amendment to the claims have been filed as follows Additional Claims

1. Other cell types (referred to on line 29 of page 5). can include the patient s own bone marrow-derived anginblasts.

2. instead of the bi1ayer of extracellular matrices, the autologous acellular mixed matrix (derived Tom dermal micro vascular endothelial cells and demlal fibroblasts) may be used on its own. This technique will be representative of transferring material from one organ of the body (the skin) to another organ (the heart) within the same human body.

Additional Claim

3. Other cell types (referred to on Zinc 2'3 of page 5), can Include the patent's own bone ' cl marrow-derived (haemo)angioblasts in a co-culture with autologous myoblasts9Ro) 0 Stretching will be optional. a+R'C