Functional Evaluations of Ex Vivo Induced Endothelial Progenitors for Autologous Transplantation in Non-Human Primates

It is possible to treat ischemia and hemophilia A diseases by producing sufficient functional human endothelial progenitor cells (EPCs)/endothelial cells (ECs) in vitro, for use with cell therapy in the clinic. We have previously reported the ability to produce FVIII-secreting EPCs/ ECs derived from human cord blood CD34⁺ cells. About 1412±102 fold expansion over initial EPCs was achieved after culturing for 21 days. An acute liver sinusoidal endothelial cells (LSEC) injury model in NOD/SCID mice was also developed to verify the functional migrating ability of the generated EPCs/ ECs in vivo.

Here, we further applied this culturing technique to expand and subsequently differentiate CD34⁺ cells into the EPCs/ ECs derived from mobilized peripheral bloods of both human and cynomolgus monkeys. In brief, the CD34⁺ cells were isolated from human peripheral bloods or from monkeys (n=10) mobilized with human G-CSF/SCF. In the first 6 days, the isolated CD34⁺ cells were expanded in modified IMDM medium supplemented with human cytokine combinations of SCF, Flt-3L, TPO, IL-3, GM-CSF, and VEGF. From days 7 to 36, the adhering EPCs/ ECs were subsequently differentiated in EBM-2 basal medium with 20% FBS and endothelial growth factors of VEGF、IGF、EGF、FGF, and fibronectin. The purities and phenotypes of the induced EPCs/ECs were assessed in vitro by antibodies against human CD31, vWF, and FVIII for the human or Dil- acetylated- low density lipoprotein (ac-LDL) and FITC-lectin double staining for the monkey cells.In addition, the safety and efficacy of the induced monkey EPCs/ECs was determined in vivo by autologous transplantation in monkey LSEC injury model, which was induced by a toxic agent, monocrotaline (MCT), to disrupt the sinusoidal endothelial barrier and stimulate the incorporation of transplanted cells into liver parenchyma. In the transplantation group (n=7), each monkey was injected with double labeled autologous EPCs/ECs preparations (2×10⁸ cells/500μl in saline), whereas in the control group (n=3) was injected with the same volume saline via hepatic portal vein injections. The cross-sections (20µm in depth) of fixed hepatic tissues were analyzed for grafting and functional migration of transplanted EPCs/ECs. The transplanted cells were identified by lenti-viral gene expressed with green fluorescent protein (red) or direct observation using anti-monkey IgG -microbead- FITC conjugates (green).

For in vitro induced EPCs/ECs derived from human peripheral blood cell, the expansion of 834.58±119.03 fold was achieved from the CD34⁺/VEGFR2⁺ EPCs on day 21. Total more than 2x 10⁸ FVIII-producing EPCs / ECs were produced from one collection of human peripheral blood (250 mL). On the other hand, the CD34⁺/VEGFR2⁺ EPCs (3.6×10⁴ ±2.1×10³) from one collection of monkey peripheral blood (20ml) were expanded up to 1274±166 fold and 7211±372 fold on days 24 and 36, respectively (n=4). The EPCs were reached at a logarithmic growth from days 12 to 45. The induced cells can be frozen and resuscitated during any stage of the culturing process. The formation of EC tubes was observed from day 24. Over 80% of expanded cells were EPC/EC-specific and identified by Dil-ac-LDL and FITC-lectin double staining on day 36. All monkeys recoveredfrom the surgeries of portal vein injection and resumed normal diet and behavior after autologous transplantation with cultured EPCs/ECs. Similarly, the routine blood analysis and liver functional enzymes were at the normal level, and no other apparent side effects were observed. About 3.2±1.4% and 2.1±1.1% of liver cells were observed as Dil-ac-LDL and FITC-lectin double positive in the liver cryosections (25 sections per monkey) on days 7 and 14, respectively, indicating that autologous transplanted EPCs/ECs were capable of repopulating into functional ECs in vivo. Furthermore, the injected EPCs/ECs were scattered in the intercellular spaces of hepatocytes at the hepatic tissues on day 14, suggesting that the transplanted cells could migrate towards injured LSEC sites and reconstitute structurally the sinusoidal endothelial compartment in monkey livers.

In summary, the large-scale EPCs/ECs were produced from CD34⁺ cells of both human and monkey peripheral bloods in vitro. The safety and functions of the EPCs/ECs were confirmed in mice and cynomolgus monkeys, strongly suggesting the potential application of these FVIII-producing EPCs/ECs to future clinical study.