Naoko Nakamura

A Feeder‐Free and Efficient Production of Functional Neutrophils from Human Embryonic Stem Cells

A novel, feeder‐free hematopoietic differentiation protocol was established for highly efficient production of neutrophils from human embryonic stem cells (hESCs). For the induction of differentiation, spheres were generated in the presence of serum and cytokine cocktail and subjected to attachment culture on gelatin‐coated plates. After approximately 2 weeks, a sac‐like structure filled with abundant round cells emerged at the center of flattened spheres. After cutting off this sac‐like structure, round cells actively proliferated, either floating in the supernatant or associated weakly with the adherent cells. Almost all of these round cells were CD45‐positive hematopoietic cells with myeloid phagocytic markers (CD33 and CD11b), and approximately 30%–50% of the round cells were mature neutrophils, as judged from morphology, cytochemical characteristics (myeloperoxidase and neutrophil alkaline phosphatase), and neutrophil‐specific cell surface markers (CD66b, CD16b, and GPI‐80). In addition, hESC‐derived neutrophils had chemotactic capacity in response to the bacterial chemotactic peptide formyl‐methionyl‐leucyl‐phenylalanine and neutrophil‐specific chemokine interleukin (IL)‐8. Using “semipurified” neutrophils migrated to IL‐8, both phagocytic and respiratory burst activities were demonstrated. Finally, it was shown that hESC‐derived neutrophils had chemotactic activity in vivo in a murine air‐pouch inflammatory model. The present results indicate successful induction of functional mature neutrophils from hESCs via highly efficient feeder‐free differentiation culture system of human hematopoietic cells. STEM CELLS 2009;27:59–67

Feeder-Free and Serum-Free Production of Hepatocytes, Cholangiocytes, and Their Proliferating Progenitors from Human Pluripotent Stem Cells: Application to Liver-Specific Functional and Cytotoxic Assays

We have established a serum- and feeder-free culture system for the efficient differentiation of multifunctional hepatocytes from human embryonic stem (ES) cells and three entirely different induced pluripotent stem (iPS) cells (including vector/transgene-free iPS cells generated using Sendai virus vector) without cell sorting and gene manipulation. The differentiation-inducing protocol consisted of a first stage; endoderm induction, second stage; hepatic initiation, and third stage; hepatic maturation. At the end of differentiation culture, hepatocytes induced from human pluripotent stem cells expressed hepatocyte-specific proteins, such as α-fetoprotein, albumin, α1 antitrypsin and cytochrome P450 (CYP3A4), at similar or higher levels compared with three control human hepatocyte or hepatic cell lines. These human iPS/ES cell-derived hepatocytes also showed mature hepatocyte functions: indocyanine green dye uptake (≈ 30%), storage of glycogen (>80%) and metabolic activity of CYP3A4. Furthermore, they produced a highly sensitive hepatotoxicity assay system for D-galactosamine as determined by the extracellular release of hepatocyte-specific enzymes. Hepatoprotective prostaglandin E1 attenuated this toxicity. Interestingly, bile duct-specific enzymes were also detected after drug treatment, suggesting the presence of bile-duct epithelial cells (cholangiocytes) in our culture system. Electron microscopic studies confirmed the existence of cholangiocytes, and an immunostaining study proved the presence of bipotential hepatoblasts with high potential for proliferation. Differentiated cells were transferrable onto new dishes, on which small-sized proliferating cells with hepatocyte markers emerged and expanded. Thus, our differentiation culture system provides mature functional hepatocytes, cholangiocytes, and their progenitors with proliferative potential from a wide variety of human pluripotent stem cells.

High-efficiency production of subculturable vascular endothelial cells from feeder-free human embryonic stem cells without cell-sorting technique.

We previously reported a feeder-free culture method for pure production of subculturable vascular endothelial cells (VECs) from cynomolgus monkey embryonic stem cells (cmESCs) without as using cell-sorting technique. By this method, canonical vascular endothelial (VE)-cadherin/platelet-endothelial cell adhesion molecule 1 (PECAM1)-positive VECs (c-VECs) and atypical VE-cadherin/PECAM1-negative VECs (a-VECs) were generated without a contamination by pericytes, lymphatic endothelial cells, or immature ES cells. More recently, we established a unique culture technique to maintain human ESCs (hESCs) under a feeder-free and recombinant cytokine-free condition. Combining these two systems, we have successfully generated pure VECs from two lines of hESCs, khES-1 and khES-3, under a completely feeder-free condition. Our method is very simple: spheres generated from hESCs by floating culture using differentiation media supplemented with vascular endothelial growth factor, bone morphogenetic protein 4, stem cell factor, FMS-related tyrosine kinase-3 ligand, and interleukin 3 (IL3) and IL6 were cultured on gelatin-coated plates. Cell passage was performed by an ordinary enzymatic treatment. The hESC-derived differentiated cells demosntrated cord-forming activities and acetylated low-density lipoprotein-uptaking capacities. Moreover, they exclusively expressed von Willebrand factor and endothelial nitric oxide synthase. Flow cytometric analyses indicate that khES-3 generated both c-VECs and a-VECs as in the case of cmESCs. By contrast, khES-1 produced only a-VECs, which nonetheless demonstrated effective recruitment into neovascularity in vivo. Interestingly, a-VECs turned to express PECAM1 after transplantation into immunodeficient mice. The hESC-derived VECs were subculturable at least up to 10 passages without functional depression. Our method does not require a presorting processes to enrich progenitor fractions such as CD34-positive or kinase insert domain receptor (KDR)-positive cells, providing the most efficient and easiest technique for VEC production from hESCs.

Highly efficient and feeder-free production of subculturable vascular endothelial cells from primate embryonic stem cells

The vascular endothelial cell (VEC) differentiation from primate embryonic stem (ES) cells has critical problems: low differentiation efficiencies (<2%) and/or subculture incapability. We report a novel feeder‐free culture method for high efficiency production of subculturable VECs from cynomolgus monkey ES cells. Spheres, which were generated from ES cells in the presence of cytokine cocktail, were cultured on gelatin‐coated plates. Cobblestone‐shaped cells spread out after a few days, which were followed by an emergence of a sac‐like structure containing hematopoietic cells. All adherent cells including sac walls cells and surrounding cobblestone cells expressed vascular endothelial cadherin (VE‐cadherin) at intercellular junctions. Subculture of these cells resulted in a generation of homogeneous spindle‐shaped population bearing cord‐forming activities and a uniform acetylated low density lipoprotein‐uptaking capacity with von Willbrand factor and endothelial nitric oxide synthetase expressions. They were freeze–thaw‐tolerable and subculturable up to eight passages. Co‐existence of pericytes or immature ES cells was ruled out. When introduced in a collagen sponge plug implanted intraperitoneally in mice, ES‐derived cells recruited into neovascularity. Although percentages of surface VE‐cadherin‐positive population varied from 20% to 80% as assessed by flow cytometry, the surface VE‐cadherin‐negative population showed intracellular VE‐cadherin expression and mature functions, as we call it as atypical VECs. When sorted, the surface VE‐cadherin‐positive population expanded as almost pure (>90%) VE‐cadherin/PECAM‐1‐positive VECs by 160‐fold after five passages. Thus, our system provides pure production of functional, subculturable and freeze–thaw‐tolerable VECs, including atypical VECs, from primate ES cells. J. Cell. Physiol. 217: 261–280, 2008.

Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells

The potential applications of human embryonic stem cells (hESCs) in regenerative medicine and developmental research have made stem cell biology one of the most fascinating and rapidly expanding fields of biomedicine. The first clinical trial of hESCs in humans has begun, and the field of stem cell therapy has just entered a new era. Here, we report seven hESC lines (SEES-1, -2, -3, -4, -5, -6, and -7). Four of them were derived and maintained on irradiated human mesenchymal stem cells (hMSCs) grown in xenogeneic-free defined media and substrate. Xenogeneic-free hMSCs isolated from the subcutaneous tissue of extra fingers from individuals with polydactyly showed appropriate potentials as feeder layers in the pluripotency and growth of hESCs. In this report, we describe a comprehensive characterization of these newly derived SEES cell lines. In addition, we developed a scalable culture system for hESCs having high biological safety by using gamma-irradiated serum replacement and pharmaceutical-grade recombinant basic fibroblast growth factor (bFGF, also known as trafermin). This is first report describing the maintenance of hESC pluripotency using pharmaceutical-grade human recombinant bFGF (trafermin) and gamma-irradiated serum replacement. Our defined medium system provides a path to scalability in Good Manufacturing Practice (GMP) settings for the generation of clinically relevant cell types from pluripotent cells for therapeutic applications.

Early Senescence Is Not an Inevitable Fate of Human-Induced Pluripotent Stem-Derived Cells

Human-induced pluripotent stem cells (hiPSCs) are expected to become a powerful tool for regenerative medicine. Their efficacy in the use of clinical purposes is currently under intensive verification. It was reported that hiPSC-derived hemangioblasts had severely limited expansion capability due to an induction of early senescence: hiPSC-derived vascular endothelial cells (VECs) senesced after one passage and hiPSC-derived hematopoietic progenitor cells (HPCs) showed substantially decreased colony-forming activities. Here we show that early senescence is not an inevitable fate of hiPSC-derived cells. Applying our unique feeder-free culture methods for the differentiations of human embryonic stem cells (hESCs), we successfully generated VECs and HPCs from three lines of hiPSCs that were established by using a retrovirus vector system. All hiPS-derived VECs could be subcultured by 2:1∼3:1 dilutions up to 10∼20 passages, after which the cells underwent senescence. Among the three lines of hiPSCs, two lines generated HPCs that bore comparable granulocyte colony-forming units to those of hESCs. Moreover, one line effectively reproduced HPCs within the sac-like structures, the fields of in vitro hematopoiesis, as in the case of hESCs. Surprisingly, release of neutrophils into culture supernatant persisted even longer (∼60 days) than the case of hESCs (∼40 days). Thus, the problem of early senescence can be overcome by selecting appropriate lines of hiPSCs and applying proper differentiation methods to them.

Human Embryonic Stem Cells with Maintenance under a Feeder-Free and Recombinant Cytokine-Free Condition

We previously reported that cynomolgus monkey embryonic stem (ES) cells could be maintained under a feeder-free condition without using recombinant cytokines if sizes and numbers of ES colonies were kept within an appropriate range. Here we show that this finding is also true with human ES cells (hESCs). The two lines of hESCs, khES-1 and khES-3, were appropriately maintained in the absence of feeder layers or exogenous cytokines such as fibroblast growth factors, Noggin, transforming growth factor beta, and Activin by closely controlling the size and number of hESC colonies. High-level expressions of immature markers including SSEA-4, Oct-4, and Nanog were detected in feeder-free and cytokine-free hESCs, and they formed teratomas when implanted into severe combined immunedeficiency (SCID) mice. No chromosomal abnormalities were observed over 20 passages, ruling out the possibility that special clones with growth advantages had been selected. Global protein expression profiles were quite similar among the hESCs maintained by our feeder- and cytokine-free method, by coculture with mouse embryonic fibroblasts (MEFs) and by a feeder-free method using conditioned media of MEFs. However, the activation level of Akt, an important player for the maintenance of ES cells, was highest and the activation level of extracellular signal-regulated kinase, a critical player for differentiation of ES cells, was lowest in the hESCs maintained by our cytokine-free method. Our results not only show a technical improvement for the maintenance of hESCs but also open a new avenue for the understanding of autocrine signaling networks of hESCs.

A feeder-free hematopoietic differentiation system with generation of functional neutrophils from feeder- and cytokine-free primate embryonic stem cells.

We have established a novel feeder- and recombinant cytokine-free culture system for the maintenance of primate embryonic stem (ES) cells along with a feeder-free hematopoietic differentiation protocol for high efficiency CD45-positive cell production. In our system, cynomolgus monkey ES cells were properly maintained in an undifferentiated state with high immature marker expressions and teratoma-producing activities. Embryoid bodies (EBs) were generated in the presence of serum and cytokine cocktail and subjected to attachment culture on gelatin-coated plates. After about 2 weeks, a sac-like structure filled with abundant round cells emerged at the center of flattened EB. Then total cells were collected and transferred onto new gelatin-coated plates, where cells were firmly attached and actively proliferated to confluence. After another few days culture, abundant floating cells were detected in the culture supernatant. These cells expressed high levels of CD45 (>90%), while adherent cells expressed low levels of CD45 (<10%). The former consisted of various differentiated stages of myeloid cells from immature myeloblasts to mature polymorphonuclear neutrophils and macrophages. Although the percentages of neutrophils varied between 10 to 20 depending on experiments, their mature phenotype was reproducibly confirmed by specific staining and functional assays. Our protocol provides the minimum essence for primate ES cell maintenance and hematopoietic differentiation that is beneficial from economical and clinical points of view.

In vivo maturation of human embryonic stem cell-derived teratoma over time

Transformation of human embryonic stem cells (hESC) is of interest to scientists who use them as a raw material for cell-processed therapeutic products. However, the WHO and ICH guidelines provide only study design advice and general principles for tumorigenicity tests. In this study, we performed in vivo tumorigenicity tests (teratoma formation) and genome-wide sequencing analysis of undifferentiated hESCs i.e. SEES-1, -2 and -3 cells. We followed up with teratoma formation histopathologically after subcutaneous injection of SEES cells into immunodeficient mice in a qualitative manner and investigated the transforming potential of the teratomas. Maturity of SEES-teratomas perceptibly increased after long-term implantation, while areas of each tissue component remained unchanged. We found neither atypical cells/structures nor cancer in the teratomas even after long-term implantation. The teratomas generated by SEES cells matured histologically over time and did not increase in size. We also analyzed genomic structures and sequences of SEES cells during cultivation by SNP bead arrays and next-generation sequencing, respectively. The nucleotide substitution rate was 3.1 × 10−9, 4.0 × 10−9, and 4.6 × 10−9 per each division in SEES-1, SEES-2, and SEES-3 cells, respectively. Heterozygous single-nucleotide variations were detected, but no significant homologous mutations were found. Taken together, these results imply that SEES-1, -2, and -3 cells do not exhibit in vivo transformation and in vitro genomic instability.