**5. Concluding remarks**

hereditary disease where benefit for patient overwhelms existing risks [101]; however, for treatment of PAD and MI, additional measures of precaution will be required prior to active clinical trials. Nevertheless, recently a group led by Dr. J. Laird began a phase I trial to evaluate the use of VEGF-expressing MSC in patients with critical limb ischemia. The trial is now ongoing with expected completion in 2017 and preclinical data indicated good safety profile with long-term expression of VEGF in MSC after viral modification [102]. Recent progress in virus biology and gene engineering allowed development of safer vector systems with controlled expression, integration, or directed insertion to genomic "safe harbors" where they induce minimal to none disturbances [103]. Preclinical evaluation of these systems is expected

Cell sheets (CS) were first introduced by Dr. Okano's group and occupied a niche between 3D tissue engineering and 2D cell cultures used to obtain therapeutic cellular materials [104]. Briefly, CS is an attached mono- or multilayered xeno-free construct that consists of viable cells with ECM produced by these cells. Application of this method allowed to circumvent a crucial setback observed in a number of experimental works—poor survival of cells used for therapeutic interventions. One of main reasons for this is procedure of detachment by proteolytic enzymes leading to disruption of ECM (along with deposited cytokines) and loss of intercellular contacts resulting in anoikis and high prevalence of cell death aggravated by passage of cells through a catheter or needle causing mechanical damage. Loss of cells implanted to the tissue by injection in suspended form is estimated as 40–75% within the first 3 days [105], while CS limits this damage to minimum keeping the cells viable after delivery and enhancing their engraftment [106]. Furthermore, ECM proteins delivered as a part of the construct are known to have a beneficial impact on regeneration and do not have toxic or immunogenic features of chemical or xenogeneic scaffolds. Generation of CS is possible from MSC, fibroblasts, EC, skeletal myoblasts, induced pluripotent stem cells and cardiomyocytes derived from them, BM cells and cardiac progenitor cells [107]—literally, any adherent cell culture after it produces enough ECM to stand mechanical manipulation [108]. CS can be used to cover a significant surface making it a good technique for superficial lesions, cardiomyoplasty and ophthalmologic and microsurgical manipulations. Numerous clinical trials are being run in Japan these years to reveal their full potential in a wide array

In relation to angiogenesis, this technique was evaluated in MI models using CS from skeletal myoblasts, AD-MSC, or cardiac progenitor cells showing their ability to generate vascularized additional layer of tissue and facilitate vascular growth in underlying tissue [110, 111]. This resulted in improved ventricular function, limited MI size and fibrosis and favorable outcomes in experimental animals. Comparative study of CS vs. injection of suspended cells showed CS to be superior in terms of most functional and histological endpoints analyzed and using a bioluminescent method, the authors reported higher survival of transplanted rat neonatal cardiomyocytes after CS delivery compared to injection [112]. Recently, clinical

to give more data on long-term impact of modification and facilitate translation.

**4. Cell sheets: minimal tissue-engineered constructs**

352 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

of disorders [109].

Overall, therapeutic angiogenesis has accumulated a "critical mass" of evidence and approaches that would allow its application in practice within the next 10–15 years expanding the capabilities of treatment. However, possibility to shift from initially used non-option or critical patients may lead to better results in clinical trials, especially in gene therapy, where numerous failures put the whole concept under question several years ago. Development of cell therapy was accompanied by a large framework of regulatory, legal, ethical and industrial work to ensure safety and patients' benefit. Number of clinical trials is growing every year and fortunately no serious evidence for adverse events or other risks for subjects' health and well-being was found up-to-date.

Therapeutic angiogenesis has become one of the pioneer methods in translational medicine and its full potential is yet to be unleashed especially in the field of *ex vivo* modification and tissue-engineered approaches to increase efficacy and ensure safety.
