Human erythropoiesis is a complex multistep process that involves the differentiation of early erythroid progenitors to mature erythrocytes. Here we show that it is feasible to differentiate and mature human embryonic stem cells (hESCs) into functional oxygen-carrying erythrocytes on a large scale (10 10 -10 11 cells/6-well plate hESCs). We also show for the first time that the oxygen equilibrium curves of the hESCderived cells are comparable with normal red blood cells and respond to changes in pH and 2,3-diphosphoglyerate. Although these cells mainly expressed fetal and embryonic globins, they also possessed the capacity to express the adult -globin chain on further maturation in vitro. Polymerase chain reaction and globin chain specific immunofluorescent analysis showed that the cells increased expression of -globin (from 0% to > 16%) after in vitro culture. Importantly, the cells underwent multiple maturation events, including a progressive decrease in size, increase in glycophorin A expression, and chromatin and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to more than 60% of the cells generating red blood cells with a diameter of approximately 6 to 8 m. The results show that it is feasible to differentiate and mature hESCs into functional oxygen-carrying erythrocytes on a large scale. (Blood. 2008;112:4475-4484) IntroductionHuman embryonic stem cells (hESCs) can be propagated and expanded in vitro indefinitely, providing a potentially inexhaustible and donorless source of cells for human therapy. Hematopoietic differentiation of hESCs has been extensively investigated in vitro, and hematopoietic precursors as well as differentiated progeny representing erythroid, myeloid, macrophage, megakaryocytic, and lymphoid lineages have been identified in differentiating hESC cultures. [1][2][3][4][5][6][7][8] Previous studies also generated primitive erythroid cells from hESCs by embryoid body formation and coculturing with stromal cells. [8][9][10] However, the efficient and controlled differentiation of hESCs into homogeneous red blood cell (RBC) populations with oxygen-carrying capacity has not been previously achieved.Mammalian erythropoiesis is a complex process that involves many steps, including the differentiation of early erythroid progenitors (burst-forming units-erythroid, BFU-E) via late erythroid progenitors (colony-forming units-erythroid, CFU-E), and finally morphologically recognizable erythroid precursors. 11 Nuclear condensation is a key event in the late stages of erythropoiesis, and enucleation is the final step in the development of mature erythrocytes, although the molecular and cellular mechanisms involved in these processes are poorly understood.Here we describe an efficient method to generate functional erythroid cells from hESCs under conditions suitable for scale-up. The cells possess oxygen-transporting capacity comparable with normal RBCs and respond to changes in pH and 2,3-diphosphoglycerate. We also show that they undergo a progressive decrea...
Interest in improving the speed of DNA analysis via capillary electrophoresis has led to efforts to integrate DNA amplification into microfabricated devices. This has been difficult to achieve since the thermocycling required for effective polymerase chain reaction (PCR) is dependent on an effective contact between the heating source and the PCR mixture vessel. We describe a noncontact method for rapid and effective thermocycling of PCR mixtures in electrophoretic chip-like glass chambers. The thermocycling is mediated through the use of a tungsten lamp as an inexpensive infrared radiation source, with cooling effected with a solenoid-gated compressed air source. With temperature ramping between 94 and 55 degrees C executed in glass microchambers as rapidly as 10 degrees C/s (heating) and 20 degrees C/s (cooling), cycle times as fast as 17 s could be achieved. Successful genomic DNA amplification was carried out with primers specific for the beta-chain of the T-cell receptor, and detectable product could be generated in a fraction of the time required with commercial PCR instrumentation. The noncontact-mediated thermocycling format was not found to be restricted to single DNA fragment amplification. Application of the thermocycling approach to both quantitative competitive PCR (simultaneous amplification of target and competitor DNA) and cycle sequencing reactions (simultaneous amplification of dideoxy terminated fragments) was successful. This sets the stage for implementing DNA thermocycling into a variety of microfabricated formats for rapid PCR fragment identification and DNA sequencing.
A device for on-line extraction and concentration of peptides from a dilute sample matrix prior to direct capillary electrophoretic analysis is described. The technique, termed solid phase extraction capillary electrophoresis (SPE-CE), can facilitate analysis of peptides in the low nanograms per milliliter range. Peptides from a sample matrix are adsorbed on a reversed phase resin (C-8 or C-18) cartridge in-line with an uncoated fused-silica capillary and subsequently released for free zone electrophoresis by injection of an organic elutant. Unlike previous designs and commercially available packed-inlet capillaries, the device is easily constructed from common laboratory materials and is applicable to a wide range of conventional instrumentation and methods. This device and method has been developed for use in our laboratory as a stand-alone preparative technique, specifically to provide a second-dimensional orthogonal separation of biologically derived HPLC fractions of peptides in a single analysis. To this end, extensive effort was required in both device construction and method development to attain the successful separations which are reported in this study. Extractions of dilute peptide mixtures from sample injections exceeding, but not limited to, 20 times (48 microL) the capillary volume with apparent recovery greater than 80% are shown. The selectivity of extraction of individual components of a very dilute peptide mixture (31 ng/mL with 280 microL of sample injected) is presented. The ability to efficiently extract the individual peptides from the sample was found to be concentration-dependent for the individual peptide components over a 1600-field dilution of a common calibration mixture of nine model peptides. Varying the injected volume of elution buffer demonstrated the importance of minimizing the amount of buffer used to desorb peptides to maximize the resolution of individual peptides. This study highlights implementation for direct SPE-CE for peptide analysis and discusses for SPE tip-induced mechanism through which reversal in electoosmotic flow occurs.
The advent of improved biomarkers promises to enhance the clinical care for patients with rheumatoid arthritis (RA) and other immune-mediated disorders. We have developed an innovative approach to broadly assess the cytokine responsiveness of human PBMCs using a multistimulant panel and multiplexed immunoassays. The objective of this study was to demonstrate this concept by determining whether cytokine profiles could discriminate RA patients according to disease stage (early versus late) or severity. A 10-cytokine profile, consisting of IL-12, CCL4, TNF-α, IL-4, and IL-10 release in response to stimulation with anti-CD3/anti-CD28, CXCL8 and IL-6 in response to CMV and EBV lysate, and IL-17A, GM-CSF, and CCL2 in response to human heat shock protein 60, easily discriminated the early RA group from controls. These data were used to create an immune response score, which performed well in distinguishing the early RA patients from controls and also correlated with several markers of disease severity among the patients with late RA. In contrast, the same 10-cytokine profile assessed in serum was far less effective in discriminating the groups. Thus, our approach lays the foundation for the development of immunologic “signatures” that could be useful in predicting disease course and monitoring the outcomes of therapy among patients with immune-mediated diseases.
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