Engineering Stem Cell Derived Tissue In a Novel 3D Perfusion Bioreactor

Matthew H. M. Lim1, I. Gabrielle M. Brons2, Karel Domansky3, Linda G. Griffith3, and Roger A. L. Pedersen2. (1) Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge, CB23RA, United Kingdom, (2) Department of Surgery, University of Cambridge, Box 202, Level E9, Addenbrooke's Hospital, Hills Road, Cambridge, cb20qq, United Kingdom, (3) Biological Engineering Division and Biotechnology Process Engineering Center, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139

Using a three-dimensional perfused flow bioreactor we differentiate murine epiblast stem cells (Brons et al, 2007) into cardiomyocytes. Differentiating the cells under physiologically relevant conditions of flow enables us to promote angiogenesis resulting in vascularised cardiac tissue.

Compared to the other organs in the body the heart has one of the poorest capacities for regeneration. As the adult human heart has only a very limited regenerative capacity, any significant heart cell loss or dysfunction due to ischemia, viral infection, or inflammation may possibly lead to the development of progressive heart failure. Congestive heart failure is currently a growing epidemic that results in significant disability and mortality while placing a heavy burden on health care systems (Cohn et al., 1997). For example, it is estimated that heart failure is responsible for more hospitalizations than all forms of cancer combined. Despite advances in pharmacological, interventional, and surgical therapeutic measures, the prognosis for heart failure patients remains poor. With chronic lack of donors limiting the number of patients who can benefit from heart transplantations, the development of new therapeutic methodologies have become imperative (Capri and Gepstein, 2006).

Zimmerman et al., (2006) stated that non terminally differentiated but electrically and functionally interconnected cardiac myocyte networks have a better chance to survive after implantation in vivo and stress the importance of culturing cells in three-dimensions to effectively engineer tissue in this manner. It has previously been shown that embryonic stem cell–derived cardiomyocytes form stable grafts following transplantation into immune-suppressed animals (Klug et al., 1996). Embryonic stem cell–derived cardiomyocytes have also been shown effectively cultured in large volume using bioreactors (Zandstra et al., 2003). Although embryonic stem cells are highly proliferative and suitable for mass production, an efficient protocol is yet to be established to ensure selective cardiomyocyte induction using these cells.

Recent advances in developmental biology have clarified the involvement of critical factors in cardiomyocyte differentiation, including bone morphogenic protein and Wnt signaling proteins, and such factors have been shown to improve the efficiency of stem cell induction (Fukuda and Yuasa 2006). Current protocols yield cardiomyocytes in sufficient quantities for most basic research needs. However, a limitation of the use of embryonic stem cells for therapeutic purposes is the inefficiency with which cardiomyocytes are generated (typically 1% of a differentiating culture). Strategies based on developmental paradigms have used directed differentiation to increase cardiogenesis (Lev, et al., 2005). These methods have had some success; however, not surprisingly, no one has been able to fully recapitulate the complex mixture of cardiogenic factors and environmental cues that efficiently induce cardiomyogenesis during embryonic development (Murray et al., 2006).

Angiogenic paracrine activity has recently been suggested as the underlying mechanism of improved cardiac function following bone marrow–derived cell transplantation into injured hearts (Kinnaird et al., 2004). The potential of endothelial precursor cells to differentiate into cardiomyocytes has been demonstrated in vitro (Badorff et al., 2003 and Yamashita et al, 2000), but not yet in vivo. Nonetheless, the potential of exogenous endothelial precursor cells to contribute to angiogenesis is well established. The blood vessel wall is inherently subjected to and affected by the pulsatile hemodynamic stimulus of blood flow within the vascular lumen, and biomechanical forces intrinsically present as a result of this hemodynamic flow are believed to play an important role in vascular development, remodeling, and lesion formation (Ghajar et al., 2006). In particular, cells lining the vascular lumen are constantly subjected to shear stress at the apical endothelial surface exerted by blood flow (Ballermann et al., 1998 and Lehoux et al., 2003). Shear stress has been recognized as an important modulator of endothelial phenotype, morphology, gene expression, and, especially, differentiation (Gloe et al., 2002 and Yamamoto et al., 2003).

Huang et al., (2005) studied the impact of shear stress on embryonic stem cells seeded into the walls of a microporous tube. After a 2-day incubation under a weak pulsatile flow they showed that [wall shear stress from -0.98 to 2.2 dyne/cm2; circumferential strain 4.6–9.6 × 104 dyne/cm2] without vascular endothelial growth factor (VEGF), embryonic stem cells in the superficial layer were regularly oriented in the direction of the pulsatial flow. The cells growing into the interstices in the deeper layer showed smooth muscle-like appearance, and were smooth muscle actin (SMA)-positive. Differentiation to two different cell types and segregation of incorporated ES cells may be simultaneously encouraged by the combination of wall shear stress and circumferential strain.

In culturing large bodies of tissue, the maximal size and thickness will critically depend on oxygen and nutrient supply. Perfusion in vitro may improve the metabolic supply and could be achieved by induction of angiogenesis or vasculogenesis (Risau, 1995 and Risau et al., 1997). Although it has been shown that engineered myocardium survives if implanted on healthy and infracted hearts, under these conditions, i.e. in the absence of immediate vascularization, engineered heart muscle must be nurtured exclusively by diffusion, which is unlikely to sufficiently support thick vascular myocardial constructs. Hence, ‘‘prevascularized'' heart muscle grafts would be preferable, on the one hand, to allow construction of complex tissues in vitro and, on the other hand, to connect the vascular bed of such grafts to the recipient circulation at the time of implantation (Zimmerman et al. 2006).

In our studies we show that culturing epiblast stem cells in our bioreactor system promotes the formation of tissue expressing VEGF Receptor 1 (Flk-1) shown by both gene expression and immunostaining. Flk-1 is a marker for endothelial cells and has been shown to be a marker for vascular progenitors as well . Thus in carrying out a cardiomyocyte differentiation protocol concurrently during the establishment of the microvessel network allows us to form vascularised cardiac tissue in addition also expressing cardiac actin, troponin and SMA. Culturing vascularised tissue enhances the transport of factors and oxygen to all the cultured tissue mass giving us the ability to culture larger volumes of tissue whilst avoiding hypoxia. This will also provide a more physiologically relevant model for planned transplantation studies.

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