Calcification after myocardial infarction is independent of amniotic fluid stem cell injection.

Ischemic heart disease remains one of the most common causes of mortality in developed countries. Recently, stem cell therapy is being considered for treating ischemic heart diseases. On the other hand, there has been evidence of chondro-osteogenic mass formation after stem cell injection in the heart. In a recent publication, Chiavegato et al. (J Mol Cell Cardiol. 42 (2007) 746-759) has suggested that amniotic fluid-derived stem (AFS) cells cause chondro-osteogenic masses in the infarcted heart. The goal of the current study was to further examine the formation of such masses, specifically, the role of AFS cells in this process. Our results confirm the presence of similar bone-like masses in the left ventricular wall of infarcted rats; however, this phenomenon occurred independent of AFS cell injection into the myocardium. Moreover, AFS cell injection did not increase the presence of chondro-osteogenic masses. Echocardiographic analysis of large infarctions in rats frequently revealed the presence of echogenic structures in the left ventricular wall. We further demonstrated a significant relationship between the infarction size and chondro-osteogenic formation and subsequent decrease in cardiac function. Collectively, our study indicates that chondro-osteogenic differentiation can take place in infarcted rat heart independent of cell injection. These results have significant implications for future design and testing of stem cell therapy for treatment of cardiac muscle diseases.

In vitro cardiomyogenic potential of human amniotic fluid stem cells.

Stem cell therapy for damaged cardiac tissue is currently limited by a number of factors, including inability to obtain sufficient cell numbers, the potential tumorigenicity of certain types of stem cells and the possible link between stem cell therapy and the development of malignant arrhythmias. In this study, we investigated whether human amniotic fluid-derived stem (hAFS) cells could be a potential source of cells for cardiac cell therapy, by testing the in vitro differentiation capabilities. Undifferentiated hAFS cells express several cardiac genes, including the transcription factor mef2, the gap junction connexin43, and H- and N-cadherin. A 24 h incubation with 5-aza-2'-deoxycytidine (5-AZA-dC) induced hAFS cell differentiation along the cardiac lineage. Evidence for this differentiation included morphological changes, upregulation of cardiac-specific genes (cardiac troponin I and cardiac troponin T) and redistribution of connexin43, as well as downregulation of the stem cell marker SRY-box 2 (sox2). When co-cultured with neonatal rat cardiomyocytes (NRCs), hAFS cells formed both mechanical and electrical connections with the NRCs. Dye transfer experiments showed that calcein dye could be transferred from NRCs to hAFS cells through cellular connections. The gap junction connexin43 likely involved in the communication between the two cell types, because 12-O-tetradecanoylphorbol 13-acetate (TPA) could partially block cellular crosstalk. We conclude that hAFS cells can be differentiated into a cardiomyocyte-like phenotype and can establish functional communication with NRCs. Thus, hAFS cells may potentially be used for cardiac cell therapy.

Parthenogenesis-derived multipotent stem cells adapted for tissue engineering applications.

Embryonic stem cells are envisioned as a viable source of pluripotent cells for use in regenerative medicine applications when donor tissue is not available. However, most current harvest techniques for embryonic stem cells require the destruction of embryos, which has led to significant political and ethical limitations on their usage. Parthenogenesis, the process by which an egg can develop into an embryo in the absence of sperm, may be a potential source of embryonic stem cells that may avoid some of the political and ethical concerns surrounding embryonic stem cells. Here we provide the technical aspects of embryonic stem cell isolation and expansion from the parthenogenetic activation of oocytes. These cells were characterized for their stem-cell properties. In addition, these cells were induced to differentiate to the myogenic, osteogenic, adipogenic, and endothelial lineages, and were able to form muscle-like and bony-like tissue in vivo. Furthermore, parthenogenetic stem cells were able to integrate into injured muscle tissue. Together, these results demonstrate that parthenogenetic stem cells can be successfully isolated and utilized for various tissue engineering applications.

Angiogenic gene modification of skeletal muscle cells to compensate for ageing-induced decline in bioengineered functional muscle tissue.

OBJECTIVE: To explore the effects of ageing on the viability of bioengineered striated muscle tissue in vivo, and if this viability can be enhanced by concurrent neovascularization, as its utility for the treatment of stress urinary incontinence (SUI) might be reduced if muscle cells are derived from old patients. MATERIALS AND METHODS: Myoblasts were obtained and expanded in culture from young (2 weeks), mature (3 months) and old (24 months) mice, and were engineered to express vascular endothelial growth factor (VEGF) to stimulate neovascularization. Myoblasts were injected subcutaneously into male nude mice and after 2 and 4 weeks, the engineered muscle tissues were harvested. RESULTS: Bioengineered muscle tissues were formed in all groups, but the engineered muscles formed by myoblasts from old mice were smaller and less contractile. However, the bioengineered muscles expressing VEGF had a greater mass and better contractility in all age groups. CONCLUSION: This pilot study showed that there was an age-related decline in the size and function of bioengineered muscle; however, there was an improvement in volume and function when the muscle cells were expressing VEGF.

Non-invasive longitudinal tracking of human amniotic fluid stem cells in the mouse heart.

Human stem cells from various sources have potential therapeutic applications. The clinical implementation of these therapies introduces the need for methods of noninvasive tracking of cells. The purpose of this study was to evaluate a high resolution magnetic resonance imaging (MRI) technique for in vivo detection and tracking of superparamagnetic micron sized iron oxide particle (MPIO)-labeled human amniotic fluid stem (hAFS) cells injected in the mouse heart. Because of the small subject size, MR signal and resolution of the in vivo MRI were increased using strong gradients, a 7.0 Tesla magnet, and an ECG and respiratory gated gradient echo sequence. MRI images of mouse heart were acquired during a 4 week course of this longitudinal study. At the end of the study, histological analysis was used to correlate cell localization with the MRI results. Introduction of MPIOs into hAFS had no significant effect upon cell proliferation and differentiation. Results of flow cytometry analysis indicated that hAFS cells remained labeled for up to 4 weeks. MRI of MPIO-labeled hAFS cells injected in agarose gels resulted in significant hypointense regions. Labeled hAFS cells injected into mouse hearts produced hypointense regions in the MR images that could be detected 24 hours and 7, 14, 21 and 28 days post injection. The co-localization of labeled cells within the hypointense regions was confirmed by histological analysis. These results indicate that high resolution MRI can be used successfully for noninvasive longitudinal tracking of hAFS cells injected in the mouse heart. The potential utility of this finding is that injected stem cells can be tracked in vivo and might serve to monitor cell survival, proliferation and integration into myocardial tissue.

Injection of autologous muscle stem cells (myoblasts) for the treatment of vocal fold paralysis: a pilot study.

OBJECTIVE: Autologous muscle stem cell (myoblast) therapy may be an ideal treatment for vocal fold paralysis because of its technical ease (administered by injection), its potential to restore muscular defects and dynamic function, and its autologous origin. The goal of this project was to determine whether autologous myoblast injection into the thyroarytenoid (TA) muscle after recurrent laryngeal nerve (RLN) injury could attenuate TA muscle atrophy and enhance spontaneous reinnervation. STUDY DESIGN: This was an animal experiment. METHODS: Unilateral RLN transection and sternocleidomastoid muscle (approximately 1 g) biopsies were performed in 16 male Wistar rats. Biopsies were used to create myoblast cultures for each animal. One month later, 10(6) autologous myoblasts labeled with fluorescent cell membrane marker (PKH26) were injected into the denervated TA of each study animal, with saline injected into controls. Animals were euthanized at 2 weeks and 2 months after myoblast injection. Outcomes included myoblast survival, TA fiber diameter and volume, and reinnervation status (motor endplate to nerve contact staining). RESULTS: All denervated TA study specimens demonstrated viable myoblasts under fluorescent microscopy, with the myoblasts demonstrating fusion with the TA myofibers at 2 months. The myoblast-treated group had greater mean TA fiber diameter than denervated TA controls at 2 months (25.1 vs. 21.1 microm; P = .04) but not at 2 weeks (25.7 microm vs. 23.5 microm; P = .06). Mean TA volumes were greater in the myoblast-treated groups at both time points. Two of the animals in the myoblast-treated group demonstrated adductor motion at 2 months, whereas none of the 2 week study animals or controls recovered adduction. Reinnervation was not significantly different between the myoblast-treated groups and the denervated controls. CONCLUSIONS: Autologous myoblast therapy may be a future treatment for vocal fold paralysis, with current findings demonstrating myoblast survival with attenuation of TA muscle atrophy.

Amniotic fluid and placental stem cells.

Human amniotic fluid has been used in prenatal diagnosis for more than 70 years. It has proven to be a safe, reliable, and simple screening tool for a wide variety of developmental and genetic diseases. However, there is now evidence that amniotic fluid may have more use than only as a diagnostic tool and may be the source of a powerful therapy for a multitude of congenital and adult disorders. A subset of cells found in amniotic fluid and placenta has been isolated and found to be capable of maintaining prolonged undifferentiated proliferation as well as able to differentiate into multiple tissue types encompassing the three germ layers. It is possible that in the near future, we will see the development of therapies using progenitor cells isolated from amniotic fluid and placenta for the treatment of newborns with congenital malformations as well as of adults, using cryopreserved amniotic fluid and placental stem cells. In this chapter, we describe a number of experiments that have isolated and characterized pluripotent progenitor cells from amniotic fluid and placenta. We also discuss various cell lines derived from amniotic fluid and placenta and future directions for this area of research.