ABSTRACT
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.
Subject(s)
Cell Separation/methods , Colony-Forming Units Assay , Lasers , Pluripotent Stem Cells/cytology , Cell Differentiation , Cell Line , Cell Survival , Humans , Karyotyping , Microdissection , PressureABSTRACT
Isolated and expanded scl (+) adult murine progenitors show a strong endothelial and hematopoietic differentiation potential and have been considered to be the adult equivalent of the hemangioblast. These unique cells may provide effective therapeutic approaches to tissue damage resulting from hypoxemia or chronic ischemia. Here, we study the fate of adult scl (+/+) during development and their ability to reverse genetic defects in scl expression. scl (+/+) adult stem cells (clone RM26) did not persist during embryonic development after injection into blastocysts of allogeneic wild-type mice on day E 3.5. However, GFP(+)-marked scl (+/+) cells were detected in all possible genotypes from allogeneic scl (+/+) intercrosses (scl (+/+), scl (+/-), scl (-/-) on day E 9.5 after the cloned cells were injected into scl-mutant blastocysts on day E 3.5. Nevertheless, there was no indication of phenotypic rescue of the mutant blastocysts despite the continued presence of scl (+/+) RM26 cells in the allogeneic embryonic environment. The results show that differentiated stem cells providing a defective gene may exert effects during development when there is a reparative demand, but they are not capable of reversing the effects of a mutant phenotype during embryonic development. These effects should be considered when evaluating the efficacy of stem cells for therapeutic reversal of inborn errors of development.
Subject(s)
Blastocyst/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Mutation , Neovascularization, Physiologic , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/physiology , Transcription Factors/genetics , Transcription Factors/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors , Blood Cells/cytology , Cell Differentiation , DNA/metabolism , Embryo, Mammalian/metabolism , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/metabolism , Hypoxia , Ischemia , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Phenotype , Stem Cells/cytology , T-Cell Acute Lymphocytic Leukemia Protein 1 , Time Factors , Transplantation, HomologousABSTRACT
Recent advances in genomics and proteomics have generated a change in emphasis from hypothesis-based to discovery-based investigations. Genomic and proteomic studies based on differential expression microarrays or comparative proteomics often provide many potential candidates for functionally important roles in normal and diseased cells. High throughput technologies to address protein and gene function in situ are still necessary to exploit these emerging advances in gene and protein discovery in order to validate these identified targets. The pharmaceutical industry is particularly interested in target validation, and has identified it as the critical early step in drug discovery. An especially powerful approach to target validation is a direct protein knockdown strategy called chromophore-assisted laser inactivation (CALI) which is a means of testing the role of specific proteins in particular cellular processes. Recent developments in CALI allow for its high throughput application to address many proteins in tandem. Thus, CALI may have applications for high throughput hypothesis testing, target validation or proteome-wide screening.
