An automated system for delivery of an unstable transcription factor to hematopoietic stem cell cultures.

An automated delivery system for cell culture applications would permit studying more complex culture strategies and simplify measures taken to expose cells to unstable molecules. We are interested in understanding how intracellular TAT-HOXB4 protein concentration affects hematopoietic stem cell (HSC) fate; however, current manual dosing strategies of this unstable protein are labor intensive and produce wide concentration ranges which may not promote optimal growth. In this study we describe a programmable automated delivery system that was designed to integrate into a clinically relevant, single-use, closed-system bioprocess and facilitate transcription factor delivery studies. The development of a reporter cell assay allowed for kinetic studies to determine the intracellular (1.4 +/- 0.2 h) and extracellular (3.7 +/- 1.8 h and 78 +/- 27 h at 37 degrees C and 4 degrees C, respectively) half-lives of TAT-HOXB4 activity. These kinetic parameters were incorporated into a mathematical model, which was used to predict the dynamic intracellular concentration of TAT-HOXB4 and optimize the delivery of the protein. The automated system was validated for primary cell culture using human peripheral blood patient samples. Significant expansion of human primitive progenitor cells was obtained upon addition of TAT-HOXB4 without user intervention. The delivery system is thus capable of being used as a clinically relevant tool for the exploration and optimization of temporally sensitive stem cell culture systems.

Comparing optic nerve-head-size measurements by the heidelberg retina tomograph with fundus photography performed with a novel focusing technique.

PURPOSE: To compare disc areas as measured with the Heidelberg Retina Tomograph (HRT) to those obtained with a fundus camera. DESIGN: A nonrandomized comparative trial. PARTICIPANTS: Forty-six patients (88 eyes) who were participating in a glaucoma-screening study. METHODS: Disc areas from fundus photographs taken with a Nidek nonmydriatic camera were measured in pixels (Adobe photoshop 7.0). A magnification factor was determined, using a previously described technique that uses the position of the focusing slide on the camera to account for the spherical refractive error. The resulting calculated disc areas were then compared with the disc areas as measured with the HRT. MAIN OUTCOME MEASURES: Disc area. RESULTS: The limits of agreement between the 2 techniques were 0.49 and -0.51 mm2, indicating that the range of differences between these imaging techniques was approximately 1 mm2. Using a Bland-Altman plot, the distribution of residuals indicated that the 2 techniques do not differ in a systematic way. CONCLUSIONS: Disc area measurements can be reasonably determined from digital fundus photography if corrected for focusing slide position when compared with HRT. This may have useful applications for both clinical glaucoma patient care and population-based glaucoma-screening programs.

Endothelial-like vascular progenitor cells (VPCs) from allogeneic and autologous donors: mobilization features distinct from hematopoietic progenitors.

Endothelial-like progenitor cells circulate in the peripheral blood (PB) and can be enumerated using cell culture-based progenitor assays. These circulating vascular progenitor cells (VPCs) are implicated in new vessel formation and regenerative potential in several animal and human models of tissue injury. Given the emerging role of VPCs in regenerative processes and the limited information on the availability of such progenitor cells, we sought to determine baseline circulating VPC levels in healthy allogeneic donors and autologous hematopoietic transplant patients. VPC numbers were also measured in peripheral blood stem cell (PBSC) grafts from both graft types. Immunohistochemistry revealed that VPC clusters obtained under our culture conditions were CD45(+) and acquired endothelial features (CD31 and vascular endothelial-cadherin) in vitro upon angiogenic stimulation and gradually lost monocytic surface markers (CD14). Before PBSC mobilization, VPCs levels varied substantially in healthy donors and were markedly lower in patients with hematologic malignancies compared with healthy allogeneic donors with 27 +/- 15 versus 99 +/- 21 VPCs/mL (mean +/- SEM), respectively (P = .001). In patients undergoing stem cell mobilization, VPCs in the PB increased from 7 +/- 2 on day 0 to 51 +/- 9 by day 7 of mobilization (P = .05), representing a median fold increase of 8.9 (range, 3.0-29.8). Although autologous transplant patients underwent more intensive mobilization, VPCs were higher in allogeneic (7.2 +/- 1.4 x 10(3)/kg) than in autologous (2.6 +/- 1.5 x 10(3)/kg) mobilized PB grafts (P = .045). To identify predictors of VPC content, graft VPCs were compared with levels of CD34(+) cells, total colony forming unit (CFU), or granulocyte-macrophage colony forming unit (GM-CFU). None of these hematopoietic progenitors correlated with VPC numbers in PBSC grafts (P = NS). However, PB monocyte levels were highly correlated with circulating VPC levels (r = 0.71, P < .0001). Thus, our analysis identified significant variability in VPCs at baseline and in PBSC grafts from healthy donors. Nevertheless, these donors remain a better source of VPCs than do autologous transplant patients. Importantly, VPC mobilization occurs independently of hematopoietic mobilization. In view of the potential role of VPCs in recovery from transplant-related tissue injury, angiogenic mobilization strategies that complement hematopoietic mobilization will need to be specifically designed.

A new fundus camera technique to help calculate eye-camera magnification: a rapid means to measure disc size.

OBJECTIVE: To find a simple means for calculating eye-camera magnification to permit estimation of true retinal object size from a retinal photograph. METHODS: The position of the focusing knob on 3 different retinal cameras (TRC-50F and TRC-50X; Topcon America Corp, Paramus, NJ; and the CR6-45NM Non-mydriatic Retinal Camera; Canon Inc, Tokyo, Japan) was measured during optic nerve photography and correlated with the refractive error, or spectacle refraction, of the subject (N = 11 for each camera). RESULTS: A strong correlation was found between focusing knob position and spectacle refraction for each of the 3 cameras tested (r = 0.96, r = 0.99, and r = 0.97, respectively). CONCLUSIONS: The focusing knob position reflects the spectacle refraction of the eye being photographed, and spectacle refraction is known to correlate well with eye-camera magnification. Therefore, focusing knob position can be used to help calculate eye-camera magnification and, hence, true retinal object size. CLINICAL RELEVANCE: The true size of the optic nerve head is important for the diagnosis of glaucoma from a retinal photograph. This technique is a simple means to calculate optic nerve head size, which may be especially useful in mass retinal photographic screening programs.