Self-contained induction of neurons from human embryonic stem cells.

BACKGROUND: Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES) cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM). However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES) cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM) could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation. CONCLUSION/SIGNIFICANCE: By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.

Pathway for differentiation of human embryonic stem cells to vascular cell components and their potential for vascular regeneration.

OBJECTIVE: We demonstrated previously that mouse embryonic stem (ES) cell-derived vascular endothelial growth factor receptor-2 (VEGF-R2)-positive cells can differentiate into both vascular endothelial cells and mural cells. This time, we investigated kinetics of differentiation of human ES cells to vascular cells and examined their potential as a source for vascular regeneration. METHODS AND RESULTS: Unlike mouse ES cells, undifferentiated human ES cells already expressed VEGF-R2, but after differentiation, a VEGF-R2-positive but tumor rejection antigen 1-60 (TRA1-60)-negative population emerged. These VEGF-R2-positive but tumor rejection antigen 1-60-negative cells were also positive for platelet-derived growth factor receptor alpha and beta chains and could be effectively differentiated into both VE-cadherin+ endothelial cell and alpha-smooth muscle actin+ mural cell. VE-cadherin+ cells, which were also CD34+ and VEGF-R2+ and thought to be endothelial cells in the early differentiation stage, could be expanded while maintaining their maturity. Their transplantation to the hindlimb ischemia model of immunodeficient mice contributed to the construction of new blood vessels and improved blood flow. CONCLUSIONS: We could identify the differentiation process from human ES cells to vascular cell components and demonstrate that expansion and transplantation of vascular cells at the appropriate differentiation stage may constitute a novel strategy for vascular regenerative medicine.

Transplantation of neural cells derived from retinoic acid-treated cynomolgus monkey embryonic stem cells successfully improved motor function of hemiplegic mice with experimental brain injury.

We induced neural cells by treating cynomolgus monkey embryonic stem (ES) cells with retinoic acid. The treated cells mainly expressed betaIIItubulin. They further differentiated into neurons expressing neurofilament middle chain (NFM) in elongated axons. Half of the cells differentiated into Islet1+ motoneurons in vitro. The monkey ES-derived neural cells were transplanted to hemiplegic mice with experimental brain injury mimicking stroke. The neural cells that had grafted into periventricular area of the mice distributed extensively over the injured cortex. Some of the transplanted cells expressed the neural stem/progenitor marker nestin 2 days after transplantation. The cells expressed markers characteristic of mature motoneurons 28 days after transplantation. Mice with the neural cell graft gradually recovered motor function, whereas control animals remained hemiplegic. This is the first demonstration that neural cells derived from nonhuman primate ES cells have the ability to restore motor function in an animal model of brain injury.

Different differentiation kinetics of vascular progenitor cells in primate and mouse embryonic stem cells.

BACKGROUND: We demonstrated that vascular endothelial growth factor receptor 2 (VEGF-R2)-positive cells derived from mouse embryonic stem (ES) cells can differentiate into both endothelial cells and mural cells to suffice as vascular progenitor cells (VPCs). Here we examined whether VPCs occur in primate ES cells and investigated the differences in VPC differentiation kinetics between primate and mouse ES cells. METHODS AND RESULTS: In contrast to mouse ES cells, undifferentiated monkey ES cells expressed VEGF-R2. By culturing these undifferentiated ES cells for 4 days on OP9 feeder layer, VEGF-R2 expression disappeared, and then reappeared after 8 days of differentiation. We then isolated these VEGF-R2-positive and vascular endothelial cadherin (VEcadherin)-negative cells by flow cytometry sorting. Additional 5-day reculture of these VEGF-R2+ VEcadherin- cells on OP9 feeder layer resulted in the appearance of platelet endothelial cell adhesion molecule-1 (PECAM1)-positive, VEcadherin-positive, endothelial nitric oxide synthase (eNOS)-positive endothelial cells. On a collagen IV-coated dish in the presence of serum, these cells differentiated into smooth muscle actin (SMA)-positive and calponin-positive mural cells (pericytes or vascular smooth muscle cells). Addition of 50 ng/mL VEGF to the culture on a collagen IV-coated dish resulted in the appearance of PECAM1+ cells surrounded by SMA+ cells. In addition, these differentiated VEGF-R2+ cells can form tube-like structures in a 3-dimensional culture. CONCLUSIONS: Our findings indicate that differentiation kinetics of VPCs derived from primate and mouse ES cells were different. Differentiated VEGF-R2+ VEcadherin- cells can act as VPCs in primates. To seek the clinical potential of VPCs for vascular regeneration, investigations of primate ES cells are indispensable.

Monkey embryonic stem cell lines expressing green fluorescent protein.

The major limitation of nonhuman primate (NHP) embryonic stem (ES) cell research is inefficient genetic modification and limited knowledge of differentiation mechanisms. A genetically modified NHP-ES cell with biomarkers, such as green fluorescent protein (GFP), that allow noninvasive monitoring of transgenic cells, is a useful tool to study cell differentiation control during preimplantation and fetal development, which also plays a crucial role in the development of cell transplantation medicine. Here we report the establishment of transgenic NHP-ES cell lines that express GFP without jeopardizing their pluripotency, which was confirmed by in vitro and in vivo differentiation. These GFP-expressing ES cells reproducibly differentiated into embryoid bodies, neural cells, and cardiac myocytes. They formed teratoma composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease (SCID) mice. GFP expression was maintained in these differentiated cells, suggesting that these cells were useful for cell transplantation experiments. Furthermore, we showed that these ES cells have the ability to form chimeric blastocysts by introducing into the early preimplantation stage NHP embryo.