BMP4-SMAD1/5/9-RUNX2 pathway activation inhibits neurogenesis and oligodendrogenesis in Alzheimer's patients' iPSCs in senescence-related conditions.

In addition to increasing ß-amyloid plaque deposition and tau tangle formation, inhibition of neurogenesis has recently been observed in Alzheimer's disease (AD). This study generated a cellular model that recapitulated neurogenesis defects observed in patients with AD, using induced pluripotent stem cell lines derived from sporadic and familial AD (AD iPSCs). AD iPSCs exhibited impaired neuron and oligodendrocyte generation when expression of several senescence markers was induced. Compound screening using these cellular models identified three drugs able to restore neurogenesis, and extensive morphological quantification revealed cell-line- and drug-type-dependent neuronal generation. We also found involvement of elevated Sma- and Mad-related protein 1/5/9 (SMAD1/5/9) phosphorylation and greater Runt-related transcription factor 2 (RUNX2) expression in neurogenesis defects in AD. Moreover, BMP4 was elevated in AD iPSC medium during neural differentiation and cerebrospinal fluid of patients with AD, suggesting a BMP4-SMAD1/5/9-RUNX2 signaling pathway contribution to neurogenesis defects in AD under senescence-related conditions.

Chondrogenic differentiation of immortalized human mesenchymal stem cells on zirconia microwell substrata.

Human mesenchymal stem cells (hMSCs) that can differentiate into chondrocytes are a potential autologous cell source for repair of damaged tissue. Current methods usually induce the formation of all three chondrocyte phenotypes, hyaline, fibrous, and elastic, without the ability to selectively induce only one of them. By controlling the size of hMSC cell clusters, it may be possible to direct differentiation more uniformly toward hyaline chondrocytes. We designed new cell culture platforms containing microwells of different diameters. The platforms and wells were composed of a zirconia ceramics substratum. hMSCs briefly adhered to the substratum before releasing and entering the microwells. The physical restraints imposed by the microwells enabled hMSC clusters to homogenously differentiate into hyaline chondrocyte-like cells. Chondrogenic aggregates in microwells expressed the hyaline chondrocyte-specific genes Col II, aggrecan (ACAN), and cartilage oligomeric protein (COMP). The cultures also produced hyaline chondrocyte-specific matrix proteins Col II and ACAN homogenously throughout the aggregates. In contrast, chondrogenesis in pellet cultures was heterogeneous with the expression of nonhyaline chondrocyte genes CD105, Col X, and Col I. In these pellet cultures, hyaline and nonhyaline chondrocyte-specific matrix proteins were distributed heterogeneously. Thus, this novel ceramic microwell substratum technology efficiently directed the differentiation of hyaline chondrocyte-like cells from hMSCs. These results indicate that there is a close relationship between hMSC cluster size regulation in the microwells and differentiation tendency. This microwell culture differentiation method will provide a valuable experimental system for both experimental and potential clinical studies.

Cryopreservation of mouse embryoid bodies.

Cryopreservation of embryonic stem (ES) cells is essential to establish them as a resource for regenerative therapy. We evaluated survival adhesion rate, cell structure, gene expression, and multipotency of frozen and thawed embryoid bodies (EBs) derived from mouse ES cells. EBs were cryopreserved using the BICELL and the Program Freezer. After one week the EBs were thawed and cultured. EBs prepared in the Program Freezer had the highest survival adhesion (Program Freezer; 55-69%, BICELL; 30-38%). Though many cells in the thawed EBs were damaged, some were not, especially those prepared in the Program Freezer. RT-PCR analysis showed that genes characteristic of the three embryonic germ layers were expressed in thawed EBs cultured for one week. EBs transplanted into mice formed teratomas consisting of cells derived from the three germ layers. In conclusion, EBs frozen in the Program Freezer had higher survival adhesion rates compared to the BICELL and formed differentiated cells characteristic of the three embryonic germ layers.

Structural differentiation, proliferation, and association of human embryonic stem cell-derived cardiomyocytes in vitro and in their extracardiac tissues.

The proliferation, structural differentiation, and capacity of association of human ES cell-derived cardiomyocytes were assessed in culture and in extracardiac graft tissues. Embryoid body (EB) outgrowths having cardiomyocytes, and their transplants in mice retroperitoneum or renal subcapsular region were analyzed mainly by immunochemistry. During the culture of EB outgrowths, colonies of cardiomyocytes grew in size exhibiting synchronized beatings. Subcellular structures of those cardiomyocytes involved in the contraction, hormone production, and intercellular integration differentiated with distinct immunoreactivity for constituent proteins/peptides. Judging from PCNA staining, proliferation potential was maintained in part for more than 70 days. In teratoma tissues on post-transplantation Day 7, cardiomyocytes maintained their integration with connexin 43 and cadherin at their junctions. They partly exhibited strong PCNA reactivity. On Day 28, large part of the cardiomyocytes lost their association, dispersing among non-cardiac cells without discernible cadherin reactivity. Proliferation potential was generally low irrespective of their tissue diversity. From these results, structural differentiation and active proliferation of human ES cell-derived cardiomyocytes occurred in vitro, maintaining their association. When developed in extracardiac tissues, however, the cardiomyocytes showed low proliferation potential and reduced cellular integration. This leads to the proposal that some procedure will be necessary to accelerate or maintain the proliferation of cardiomyocytes in vivo.

Natriuretic peptides in ectopic myocardial tissues originating from mouse embryonic stem cells.

In a previous report we described the survival and contractile function of mouse embryonic stem cell-derived cardiomyocytes in the host retroperitoneum. To further understand the nature of embryonic stem cell-derived cardiomyocytes, the study assessed the synthesis of natriuretic peptides in ectopic myocardial tissues of embryonic stem cell origin. Cardiomyocytes formed in embryoid body outgrowths were transplanted into the retroperitoneum of adult nude mice, and the myocardial tissues that developed were characterized by RT-PCR and immunohistochemistry concerning atrial and brain natriuretic peptides (ANP, BNP). In the outgrowths of embryoid bodies in vitro, gene expression of ANP and BNP was detected by RT-PCR and granules positive for the peptides were identified in a few cardiomyocytes by light and electron microscopic immunocytochemistry. Seven days after transplantation the transplants exhibited multidifferentiated teratoma tissues. Developing chamber myocardial tissues positive for cardiac troponin I, cadherin, and connexin 43 were evident in the transplants, which contained ANP-positive cardiomyocytes. Transplants with beating bundles were observed 30 days after transplantation, in which gene expression of both natriuretic peptides was detected. Myocardial tissues with abundant ANP-immunoreactivity, as well as with BNP-immunoreactivity to a lesser extent, were evident in the transplants. Also, myocardial tissues without immunoreactivity for natriuretic peptides were observed. Immunoelectron microscopy showed discernible secretory granules containing ANP and/or BNP in the cardiomyocytes. These results showed that part of the cardiomyocytes in embryonic stem cell-derived ectopic myocardial tissues are capable of producing natriuretic peptides, which suggests that they may be used as an endocrine source for cardiac hormones.

Phenotype-specific cells with proliferative potential are produced by polyethylene glycol-induced fusion of mouse embryonic stem cells with fetal cardiomyocytes.

Because cardiomyocytes lose the ability to divide upon differentiation, myocardial failure is assumed to be generally irreversible. For terminal cardiac insufficiency, the potential for regenerative treatment by stem cells, especially embryonic stem (ES) cells, offers hope for the future. Recent studies showed that stem cells fuse spontaneously with cells remaining in damaged tissues, and restore tissue function. To imitate spontaneous fusion in vivo, we used polyethylene glycol (PEG) in vitro to fuse mouse ES cells and fetal cardiomyocytes and analyzed the cytochemical properties of the fused cells. Confocal laser scanning microscopy coupled with lipophilic dye labeling of the living cell membranes showed that there were fused cells of ES cells and cardiomyocytes after PEG treatment. By flow cytometry, the fusion efficiency between ES cells and cardiomyocytes was estimated to be about 45% of the total resulting cells. When green fluorescent protein (GFP)-expressing ES cells were fused with cardiomyocytes, the fused cells had immunoreactivity for GFP in their cytoplasm and cardiac troponin I in their myofibrils. Some of these cells also expressed proliferating cell nuclear antigen up to 11 days after fusion, the last time point examined. This study shows that PEG-induced fusions of mouse ES cells and cardiomyocytes have the cardiomyocyte phenotype and proliferation potential.