Automated selection and harvesting of pluripotent stem cell colonies.

The ability of pluripotent stem cells to differentiate into specialized cells of all three germ layers, their capability to self-renew, and their amenability to genetic modification provide fascinating prospects for the generation of cell lines for biomedical applications. Therefore, stem cells must increasingly suffice in terms of industrial standards, and automation of critical or time-consuming steps becomes a fundamental prerequisite for their routine application. Cumbersome manual picking of individual stem cell colonies still represents the most frequently used method for passaging or derivation of clonal stem cell lines. Here, we explore an automated harvesting system (CellCelector™) for detection, isolation, and propagation of human embryonic stem cells (hESCs) and murine induced pluripotent stem cells (iPSCs). Automatically transferred hESC colonies maintained their specific biological characteristics even after repeated passaging. We also selected and harvested primary iPSCs derived from mouse embryonic fibroblasts expressing the green fluorescent protein (GFP) under the control of the Oct4 promotor using either morphological criteria or GFP fluorescence. About 80% of the selected and harvested primary iPSC colonies gave rise to homogenously GFP-expressing iPSC lines. To validate the iPSC lines, we analyzed the expression of pluripotency-associated markers and multi-germ layer differentiation potential in vitro. Our data indicate that the CellCelector™ technology enables efficient identification and isolation of pluripotent stem cell colonies at the phase contrast or fluorescence level.

Laser-assisted photoablation of human pluripotent stem cells from differentiating cultures.

Due to their pluripotency and their self-renewal capacity, human pluripotent stem cells (hPSC) provide fascinating perspectives for biomedical applications. In the long term, hPSC-derived tissue-specific cells will constitute an important source for cell replacement therapies in non-regenerative organs. These therapeutic approaches, however, will critically depend on the purity of the in vitro differentiated cell populations. In particular, remaining undifferentiated hPSC in a transplant can induce teratoma formation. In order to address this challenge, we have developed a laser-based method for the ablation of hPSC from differentiating cell cultures. Specific antibodies were directed against the hPSC surface markers tumor related antigen (Tra)-1-60 and Tra-1-81. These antibodies, in turn, were targeted with nanogold particles. Subsequent laser exposure resulted in a 98,9 +/- 0,9% elimination of hPSCs within undifferentiated cell cultures. In order to study potential side effects of laser ablation on cells negative for Tra-1-60 and Tra-1-81, hPSC were mixed with GFP-positive hPSC-derived neural precursors (hESCNP) prior to ablation. These studies showed efficient elimination of hPSC while co-treated hESCNP maintained their normal proliferation and differentiation potential. In vivo transplantation of treated and untreated mixed hPSC/hESCNP cultures revealed that laser ablation can dramatically reduce the risk of teratoma formation. Laser-assisted photothermolysis thus represents a novel contact-free method for the efficient elimination of hPSC from in vitro differentiated hPSC-derived somatic cell populations.

Laser-assisted selection and passaging of human pluripotent stem cell colonies.

The derivation of somatic cell products from human embryonic stem cells (hESCs) requires a highly standardized production process with sufficient throughput. To date, the most common technique for hESC passaging is the manual dissection of colonies, which is a gentle, but laborious and time-consuming process and is consequently inappropriate for standardized maintenance of hESC. Here, we present a laser-based technique for the contact-free dissection and isolation of living hESCs (laser microdissection and pressure catapulting, LMPC). Following LMPC treatment, 80.6+/-8.7% of the cells remained viable as compared to 88.6+/-1.7% of manually dissected hESCs. Furthermore, there was no significant difference in the expression of pluripotency-associated markers when compared to the control. Flow cytometry revealed that 83.8+/-4.1% of hESCs isolated by LMPC expressed the surface marker Tra-1-60 (control: 83.9+/-3.6%). In vitro differentiation potential of LMPC treated hESCs as determined by embryoid body formation and multi-germlayer formation was not impaired. Moreover, we could not detect any overt karyotype alterations as a result of the LMPC process. Our data demonstrate the feasibility of standardized laser-based passaging of hESC cultures. This technology should facilitate both colony selection and maintenance culture of pluripotent stem cells.

Automated maintenance of embryonic stem cell cultures.

Embryonic stem cell (ESC) technology provides attractive perspectives for generating unlimited numbers of somatic cells for disease modeling and compound screening. A key prerequisite for these industrial applications are standardized and automated systems suitable for stem cell processing. Here we demonstrate that mouse and human ESC propagated by automated culture maintain their mean specific growth rates, their capacity for multi-germlayer differentiation, and the expression of the pluripotency-associated markers SSEA-1/Oct-4 and Tra-1-60/Tra-1-81/Oct-4, respectively. The feasibility of ESC culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.

A Korean kindred with autosomal dominant nocturnal frontal lobe epilepsy and mental retardation.

BACKGROUND: A Korean family had distinctive clinical and neuroimaging features and carried the same genetic mutation that was found in a previously described Japanese kindred with autosomal dominant nocturnal frontal lobe epilepsy. OBJECTIVE: To describe the first Korean family with autosomal dominant nocturnal frontal lobe epilepsy. METHODS: Members of a large family, including 9 affected individuals from 3 generations, underwent a comprehensive genetic, clinical, electroencephalographic, neuropsychological, and neuroimaging evaluation. Affected members were tested for possible mutations in transmembrane regions 1 through 3 of the neuronal nicotinic acetylcholine receptor alpha4 subunit (CHRNA4) by direct sequencing and subsequent restriction analysis. RESULTS: Seizures began in childhood, presenting as nocturnal episodes of staring, confusion, shouting, perioral movements, unintelligible speech, and hand waving. Some patients had ictal or interictal epileptiform activity in the temporal and/or frontocentral areas. Neurological examination and brain magnetic resonance imaging results showed no abnormalities, except that all patients available for testing had mild to moderate mental retardation. Fluorodeoxyglucose F 18 with positron emission tomography showed mild decreased glucose uptake in the superior and middle frontal regions, more so on the left than on the right. Patient response to carbamazepine was poor. All affected members were heterozygous for the CHRNA4 Ser252Leu mutation. CONCLUSIONS: Disorders associated with mutations in the transmembrane region 2 of CHRNA4 are genetically and phenotypically heterogeneous. Distinctive features of this kindred include (1) mental retardation in all affected members available for testing, (2) abnormal brain findings on fluorodeoxyglucose F 18 with positron emission tomography, (3) poor response to carbamazepine, and (4) full penetrance.

Congenital myasthenic syndrome with episodic apnea in patients homozygous for a CHAT missense mutation.

BACKGROUND: The syndrome of congenital myasthenia with episodic apnea (CMS-EA) was previously found to be due to mutations in the choline acetyltransferase gene (CHAT). OBJECTIVE: To identify the mutations underlying CMS-EA in a Turkish multiplex family. DESIGN: Direct sequencing of the CHAT gene. PATIENTS: A consanguineous Turkish family with 2 siblings affected by muscular weakness and episodic respiratory distress. RESULTS: The sequencing of CHAT coding exons identified a previously unknown missense mutation that affected a highly conserved amino acid residue (I336T). The mutation was absent in 164 control chromosomes. CONCLUSIONS: The high degree of conservation in different species strongly suggests that I336T is a functionally important amino acid residue. The absence of I336T from a large control sample further supports the pathogenic role of I336T in CMS-EA. This is the second report of CHAT mutations causing presynaptic CMS.