**6. Ability of iPSC-ECs to Induce In Vivo Neovascularization**

Over the last decade, the behavior of iPSC-ECs in vivo has been investigated in numerous studies. Already in 2011, Li et al. revealed the ability of hiPSC-ECs to form functional blood vessels by means of tissue-engineered constructs through a Matrigel plug assay performed in immunodeficient Severe Combined Immunodeficient (SCID) mice [97]. The authors isolated endothelial cells from undifferentiated hiPSCs cultured on Matrigel-coated plates that were placed into Petri dishes with differentiation medium. Then, two weeks after the subcutaneous injection of the plugs, they harvested them and performed histological analyses revealing the presence of microvessels containing murine blood cells into their lumen [97]. In order to decrease the risk of teratoma development after implantation, Margariti et al., in 2012, generated partially induced pluripotent stem cells (PiPSC) that clearly showed the ability to differentiate into endothelial cells thanks to specific culture media and conditions. To test vessel patency and perfusion the authors used an ischemic model in SCID mice to which they injected subcutaneously a mix of PiPSC-ECs and Matrigel [98]. Fourteen days after surgery, the authors harvested the plugs and compared PiPSC-ECs with both controls (no cells) and fibroblasts reporting a significantly higher blood flow displayed by the PiPSC group. Moreover, high capillary number, stained with CD31, and a typical vascular architecture were observed in engrafted PiPSC-Ecs compared to the control group, in which the injected fibroblasts formed a random pattern. Finally, the engraftment ability was reported to be improved by using PiPSC-ECs [98]. In another very interesting study, Rufaihah et al. evaluated hiPSC-EC heterogeneity [99]. In particular, they investigated whether these cells could be characterized for each subtype. They obtained all the three

principal subtypes by using different concentrations of VEGF and highlighted that arterial and venous hiPSC-ECs cytotypes were predominant. The authors injected subcutaneously into the mid lower abdominal region of SCID mice: Matrigel and bFGF (basic Fibroblast Growth Factor), heterogeneous hiPSC-ECs in Matrigel and bFGF, arterial enriched hiPSC-ECs in Matrigel and bFGF. After 14 days, Matrigel plugs were removed and immunostained with anti-CD31 Ab. Matrigel implants including hiPSC-artECs showed the ability to establish a more extensive vascular network also confirming its human origin through the positivity to human anti-CD31 immunostaining. The same work also demonstrated the onset of a widespread capillary network, especially for the arterial lineage derived from PiPSCs [99]. To further investigate and promote the use of iPSC-ECs in tissue engineering and regeneration, Clayton et al. evaluated the behavior of these cells in comparison to iECs in a mouse hind limb ischemia model. During the surgery, they made an intramuscular injection of either 1 <sup>×</sup> 106 iPSC-ECs or 1 <sup>×</sup> 106 iECs [92]. In particular, each treatment was divided into two injections of 25 <sup>μ</sup>l (for a total volume of 50 μL of solution) on each side of the adductor muscle, nearby the area where femoral vessels had been ligated and removed. Afterwards, the authors recorded perfusion data after 0, 1, 2, 4, 7, 10 and 14 days. According to Rufaihah, at day 14 post-injection, blood perfusion was notably increased in mice receiving iPSC-ECs, although in those injected with iECs the enhancement reported at all-time points also demonstrated a high pro-angiogenic response in the short-term, specifically at day 10. Moreover, this work shows that iPSC-ECs and iECs, at day 14, were integrated with the host vasculature. Finally, at the same time-point, iPSC-ECs implanted mice did not exhibit functional increasing of capillary density in the ischemic gastrocnemius muscle with respect to mice treated with iECs [92]. Another work by Tan et al. reported that iPSCs-ECs display a longer lifetime and a higher proangiogenic function when seeded on scaffolds than when administered alone [100].

The authors injected FVB/n mice subcutaneously with: control EBM media, iPSC-ECs, iPSC-EC-seeded scaffolds and scaffolds alone. Scaffolds were composed of poly-caprolactone (PCL) and gelatin. In particular, the authors used scaffolds with a PCL:gelatin ratio of 70:30 (PG73) that supported elevated levels of iPSC-ECs growth. Indeed, by injecting iPSC-ECs seeded on PG73 scaffolds the survival of these cells increased up to 3 days.

Therefore, there was an increase of the total engraftment ability of iPSC-ECs seeded on PG73 in comparison to these cells alone. Finally, it was highlighted a higher degree of blood perfusion when the cells were included into the scaffold [100]. A further significant advance about the capacity to obtain a functional microvasculature from iPSCs has been recently made by Bezenah et al., who compared iPSC-ECs to HUVECs by co-injecting subcutaneously endothelial cells (iPSC-EC or HUVEC) and human lung fibroblasts (NHLFs) included into a fibrin matrix in CB17/SCID mice [101].

iPSC-ECs showed a consistent decrease both in vessel density and number of perfused vessels compared to HUVECs. These cells were able to form patent and perfusable vessels, showing many morphological features comparable to those expressed by HUVECs. In fact, 4, 7 or 14 days after cell injection, the authors demonstrated that iPSC-ECs/NHLF fibrin implants were able to induce vascular morphogenesis, while at days 7 and 14, the constructs exhibited increased vessel diameter and perfusion, thus confirming a high degree of integration with the host vasculature. Furthermore, vessel density increased over time in the group injected with iPSC-ECs compared to the one injected with HUVECs with a peaking value at day 7 and a decrease by day 14 [101]. Furthermore, Foster et al. used ischemic NOD-SCID mice to investigate how to control early decline viability that normally happens in iPSC-ECs after implantation [102]. The authors developed an injectable, recombinant hydrogel for cell transplantation termed SHIELD (Shear-thinning Hydrogel for Injectable Encapsulation and Long-term Delivery) able to reduce cell membrane damage during injection.

They performed intramuscular injections into the gastrocnemius muscle in the following groups of animals: PBS, SHIELD 2.5, iPSC-ECs in PBS solution and iPSC-ECs in SHIELD 2.5 They have chosen SHIELD 2.5, 2.5 wt% PNIPAM, because this formulation allows cells to proliferate over 14 days.

Animals were euthanized 14 days post-treatment and the authors demonstrated that treatment with iPSC-ECs delivered within SHIELD-2.5 resulted in significantly greater arteriole density and improved formation of large microvessels, features that usually play a prominent role in neovascularization [102]. In 2019, Ye et al. focused their attention on the exosomes derived from human iPSC-ECs (hiPSC-EC-Exo) [103]. Exosomes are vesicles containing miRNAs and are able to protect them from RNAases but they also release miRNAs which are highly involved in the regulation of angiogenesis. Indeed, it has been recently demonstrated that paracrine factors obtained from iPSCs transplantation are more effective than iPSCs themselves. Therefore, Ye et al. evaluated the role of hiPSC-EC-Exo in promoting angiogenesis in a mouse model of Peripheral Artery Disease (PAD) [103]. Immediately after the ligation of mice femoral artery, the authors injected intramuscularly either PBS or exosomes (hiPSC-EC-Exo and inhibitory-miR199b-5p-Exo) by direct injection of a total volume of 20 μL into four different sites of the ischemic hind limb. Blood perfusion was monitored at day 0, 7, 14 and 21 post hiPSC-EC-Exo treatment and an additional treatment was performed twice a week thereafter. An increased blood perfusion of ischemic limbs was shown from day 14 onwards. Then, after harvesting muscle tissue, Ye et al., by showing an increased number of CD31 positive cells, demonstrated the enhancement of neovascularization with hiPSC-EC-Exo treatment respect to the vehicle (PBS) and to inhibitory- hiPSC-EC-Exo [103]. In conclusion, to contrast several ischemic pathologies, iPSC-ECs use can bypass the shortage and the low functionality of autologous stem cells in creating a vascular network able to promote tissue regeneration. In addition, iPSC-ECs can be generated in large quantities, because they do not derive from embryos and display minimal immunogenicity. Undoubtedly, the formation of teratomas is a real risk that may result from the use of these cells; however, an approach to reduce this issue can be represented by the one proposed in the work of Margariti who created "partial-iPSC-ECs" that, during differentiation, showed reduced capacity to form teratomas [98].

Finally, the approach related to the integration of scaffolds with iPSC-ECs is very promising, especially for the capacity of the scaffolds to retain cells, implying that a lower number of cells is required to improve new vessel formation in ischemic tissues.

#### **7. Conclusions**

The opportunity to produce human pluripotent cells (hiPSCs) from somatic cells is one of the most exciting breakthroughs of this scientific era. HiPSCs are useful to develop patient-specific drug screening and validation methodologies as well as to model human diseases, thus allowing to shape an individualized cell therapy. In view of this, these cells clearly represent the launching pad for an efficient personalized medicine.

First-in-Human (FIH) test have been performed in 2014 to treat age-related macular degeneration [104]. Mandai et al. transplanted an autologous iPSC-derived retinal pigment epithelium (RPE) cell sheet [104]. The study involved two patients. Patient 1 did not show any serious adverse event 25 months after implantation, meaning that the procedure did not trigger the host immune response, nor did it trigger tumor formation [104]. Patient 2 did not complete the procedure due to the detection of deletions in chromosome X patient derived iPSCs. Although tumorigenicity has never been reported in association to these deletions, the team decided to exclude patient 2 from the trial. In any case, the same cells implanted in mice did not develop teratomas. It is evident that iPSCs carry on intrinsic critical points that should be carefully analyzed. Reprogramming itself can lead to genetic and epigenetic dysregulation. As previously mentioned, some reprogramming factors are potent oncogenes [105], and it has been widely reported that the reactivation of these genes is able to cause teratoma formation. Another significant issue that should be addressed is the removal from transplanted iPSC of those cells that are not completely differentiated [106], thus implying a careful selection and an accurate screening of the cells. In this respect, in vivo teratoma assay is still an expensive and time-consuming procedure. A molecular approach based on Quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR) could be useful holding the sensitivity needed to detect undifferentiated cells [107]. More in detail, this assay relies on the detection of Lin28, a pluripotency-associated gene, used to recognize undifferentiated cells.

In conclusion, in this work, we reviewed the potential hold by hIPSC-ECs in providing an efficient alternative to primary cells for regenerative medicine applications. The use of IPSC-EC as vascular forming cells is encouraged by several studies involving the comparison with well-established cell lines both in vitro and in vivo. Due to their abilities, it is not difficult to imagine a widespread use of iPSC-EC as the preferential source of endothelial cells in tissue engineering. However, the main issue concerning the safety of their use in the clinic persists. In view of this, to fully exploit hiPSC-ECs potential, it is mandatory to set up reliable methods for their production in order to fulfill clinical grade requirements. The current protocols to induce pluripotency, guide the cells towards a mature phenotype and effectively select the resulting cells seem to be ready to go from bench to clinical trials [108].

**Author Contributions:** Conceptualization, C.A. and A.P.; methodology, C.A. and A.P.; validation, C.A., A.P. and I.M.; investigation, A.P and I.M.; resources, P.d.G.; data curation, C.L. and E.D.F.; writing—original draft preparation, A.P., I.M. and C.P.; writing—review and editing, C.A.; supervision, C.A., P.d.G. and P.A.N.; project administration, P.A.N.

**Conflicts of Interest:** The authors declare no conflict of interest.
