[Show abstract][Hide abstract] ABSTRACT: There are potential advantages to the low-temperature (-196°C) banking of isolated islets, including the maintenance of viable islets for future research. We therefore assessed the in vitro and in vivo function of islets cryopreserved for nearly 20 years.
Human islets were cryopreserved from 1991 to 2001 and thawed between 2012 and 2014. These were characterised by immunostaining, patch-clamp electrophysiology, insulin secretion, transcriptome analysis and transplantation into a streptozotocin (STZ)-induced mouse model of diabetes.
The cryopreservation time was 17.6 ± 0.4 years (n = 43). The thawed islets stained positive with dithizone, contained insulin-positive and glucagon-positive cells, and displayed levels of apoptosis and transcriptome profiles similar to those of freshly isolated islets, although their insulin content was lower. The cryopreserved beta cells possessed ion channels and exocytotic responses identical to those of freshly isolated beta cells. Cells from a subset of five donors demonstrated similar perifusion insulin secretion profiles pre- and post-cryopreservation. The transplantation of cryopreserved islets into the diabetic mice improved their glucose tolerance but did not completely normalise their blood glucose levels. Circulating human insulin and insulin-positive grafts were detectable at 10 weeks post-transplantation.
We have demonstrated the potential for long-term banking of human islets for research, which could enable the use of tissue from a large number of donors with future technologies to gain new insight into diabetes.
[Show abstract][Hide abstract] ABSTRACT: The formation of cells into more physiologically relevant three-dimensional multicellular aggregates is an important technique for the differentiation and manipulation of stem cells and their progeny. As industrial and clinical applications for these cells increase, it will be necessary to execute this procedure in a readily scalable format. We present here a method employing microwells to generate large numbers of human pluripotent stem cell aggregates and control their subsequent differentiation towards a cardiac fate.
No preview · Article · Jul 2014 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.
Full-text · Article · Nov 2013 · Journal of Visualized Experiments
[Show abstract][Hide abstract] ABSTRACT: Complex tissues contain multiple cell types that are hierarchically organized within morphologically and functionally distinct compartments. Construction of engineered tissues with optimized tissue architecture has been limited by tissue fabrication techniques, which do not enable versatile microscale organization of multiple cell types in tissues of size adequate for physiological studies and tissue therapies. Here we present an 'Intaglio-Void/Embed-Relief Topographic molding' method for microscale organization of many cell types, including induced pluripotent stem cell-derived progeny, within a variety of synthetic and natural extracellular matrices and across tissues of sizes appropriate for in vitro, pre-clinical, and clinical studies. We demonstrate that compartmental placement of non-parenchymal cells relative to primary or induced pluripotent stem cell-derived hepatocytes, compartment microstructure, and cellular composition modulate hepatic functions. Configurations found to sustain physiological function in vitro also result in survival and function in mice for at least 4 weeks, demonstrating the importance of architectural optimization before implantation.
Full-text · Article · May 2013 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation, and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and, with quantitative cell division tracking and fate monitoring, identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion, during directed differentiation, to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system.
Full-text · Article · Apr 2012 · Biotechnology and Bioengineering
[Show abstract][Hide abstract] ABSTRACT: Although it has been observed that aggregate size affects cardiac development, an incomplete understanding of the cellular mechanisms underlying human pluripotent stem cell-derived cardiomyogenesis has limited the development of robust defined-condition cardiac cell generation protocols. Our objective was thus to elucidate cellular and molecular mechanisms underlying the endogenous control of human embryonic stem cell (hESC) cardiac tissue development, and to test the hypothesis that hESC aggregate size influences extraembryonic endoderm (ExE) commitment and cardiac inductive properties. hESC aggregates were generated with 100, 1000, or 4000 cells per aggregate using microwells. The frequency of endoderm marker (FoxA2 and GATA6)-expressing cells decreased with increasing aggregate size during early differentiation. Cardiogenesis was maximized in aggregates initiated from 1000 cells, with frequencies of 0.49±0.06 cells exhibiting a cardiac progenitor phenotype (KDR(low)/C-KIT(neg)) on day 5 and 0.24±0.06 expressing cardiac Troponin T on day 16. A direct relationship between ExE and cardiac differentiation efficiency was established by forming aggregates with varying ratios of SOX7 (a transcription factor required for ExE development) overexpressing or knockdown hESCs to unmanipulated hESCs. We demonstrate, in a defined, serum-free cardiac induction system, that robust and efficient cardiac differentiation is a function of endogenous ExE cell concentration, a parameter that can be directly modulated by controlling hESC aggregate size.
Full-text · Article · Mar 2011 · Tissue Engineering Part A
[Show abstract][Hide abstract] ABSTRACT: Biomaterials are increasingly being used to engineer the biochemical and biophysical properties of the extracellular stem cell microenvironment in order to tailor niche characteristics and direct cell phenotype. To date, stem cell-biomaterial interactions have largely been studied by introducing stem cells into artificial environments, such as 2D cell culture on biomaterial surfaces, encapsulation of cell suspensions within hydrogel materials, or cell seeding on 3D polymeric scaffolds. In this study, microparticles fabricated from different materials, such as agarose, PLGA and gelatin, were stably integrated, in a dose-dependent manner, within aggregates of pluripotent stem cells (PSCs) prior to differentiation as a means to directly examine stem cell-biomaterial interactions in 3D. Interestingly, the presence of the materials within the stem cell aggregates differentially modulated the gene and protein expression patterns of several differentiation markers without adversely affecting cell viability. Microparticle incorporation within 3D stem cell aggregates can control the spatial presentation of extracellular environmental cues (i.e. soluble factors, extracellular matrix and intercellular adhesion molecules) as a means to direct the differentiation of stem cells for tissue engineering and regenerative medicine applications. In addition, these results suggest that the physical presence of microparticles within stem cell aggregates does not compromise PSC differentiation, but in fact the choice of biomaterials can impact the propensity of stem cells to adopt particular differentiated cell phenotypes.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jun 2009 · Biotechnology and Bioengineering
[Show abstract][Hide abstract] ABSTRACT: Human pluripotent cells such as human embryonic stem cells (hESC) are a great potential source of cells for cell-based therapies; however, directing their differentiation into the desired cell types with high purity remains a challenge. The stem cell microenvironment plays a vital role in directing hESC fate and we have previously shown that manipulation of colony size in a serum- and cytokine-free environment controls self-renewal and differentiation toward the extraembryonic endoderm lineage. Here we show that, in the presence of bone morphogenetic protein 2 and activin A, control of colony size using a microcontact printing technology is able to direct hESC fate to either the mesoderm or the endoderm lineage. Large, 1200-mum-diameter colonies give rise to mesoderm, while small 200-mum colonies give rise to definitive endoderm. This study links, for the first time, cellular organization to pluripotent cell differentiation along the mesoderm and endoderm lineages.
[Show abstract][Hide abstract] ABSTRACT: We report an ultra-rapid prototyping technique for forming microchannel networks for lab-on-a-chip applications, called masters on-demand. Channels are produced by replica molding on masters formed by laser printing on flexible copper printed circuit board (PCB) substrates. Masters of various designs and dimensions can be individually or mass produced in less than 10 minutes. Using this technique, we have fabricated channels as narrow as 100 microm with heights ranging between 9 microm and 70 microm. Multi-depth channel fabrication is also reported, using a two-step printing process. The functionality of devices formed in this manner is verified by performing in-channel electrophoretic separations and culture and analysis of primary mammalian cells.
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem cells (hESC) should enable novel insights into early human development and provide a renewable source of cells for regenerative medicine. However, because the three-dimensional hESC aggregates [embryoid bodies (hEB)] typically employed to reveal hESC developmental potential are heterogeneous and exhibit disorganized differentiation, progress in hESC technology development has been hindered.
Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation, we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions. These aggregates exhibited coordinated bi-domain structures including contiguous regions of extraembryonic endoderm- and epiblast-like tissue. A silicon wafer-based microfabrication technology was used to generate surfaces that permit the production of hundreds to thousands of hEB per cm(2).
The mechanisms of early human embryogenesis are poorly understood. We report an ultra high throughput (UHTP) approach for generating spatially and temporally synchronised hEB. Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis. These results should advance fundamental studies into early human developmental processes, enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues, and accelerate the pre-clinical evaluation of hESC-derived cells.
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem cells (hESCs) are an important tool for the study of developmental biology and may one day serve as a source of cells for regenerative medicine. As no definitive assay for hESC pluripotency is available, surrogate assays that measure markers or properties that have been correlated with hESC developmental potential are used to measure the effects of test conditions on their propagation and differentiation. This unit presents a range of protocols, including visual inspection, flow cytometry, immunofluorescence, quantitative real-time reverse-transcriptase PCR, and a colony-forming assay, as tools to measure the undifferentiated hESC state. The authors discuss the advantages and limitations of the various protocols, and present expected results and discuss potential problems. The development of quantitative assays of hESC developmental potential are critical for our understanding of hESC biology.
Full-text · Article · Sep 2007 · Current protocols in stem cell biology
[Show abstract][Hide abstract] ABSTRACT: Human cells appear exquisitely sensitive to the levels of hTERT expression, the telomerase reverse transcriptase. In primary cells that do not express hTERT, telomeres erode with each successive cell division, leading to the eventual loss of telomere DNA, an induction of a telomere DNA damage response, and the onset of cellular senescence or crisis. In some instances, an average of less than one appropriately spliced hTERT transcript per cell appears sufficient to restore telomerase activity and telomere maintenance, and overcome finite replicative capacity.
To underscore this sensitivity, we showed that a widely used system of transcriptional induction involving ecdysone (muristerone) led to sufficient expression of hTERT to immortalize human fibroblasts, even in the absence of induction. To permit tightly regulated expression of hTERT, or any other gene of interest, we developed a method of transcriptional control using an invertible expression cassette flanked by antiparallel loxP recombination sites. When introduced into human fibroblasts with the hTERT cDNA positioned in the opposite orientation relative to a constitutively active promoter, no telomerase activity was detected, and the cell population retained a mortal phenotype. Upon inversion of the hTERT cDNA to a transcriptionally competent orientation via the action of Cre recombinase, cells acquired telomerase activity, telomere DNA was replenished, and the population was immortalized. Further, using expression of a fluorescent protein marker, we demonstrated the ability to repeatedly invert specific transcripts between an active and inactive state in an otherwise isogenic cell background.
This binary expression system thus provides a useful genetic means to strictly regulate the expression of a given gene, or to control the expression of at least two different genes in a mutually exclusive manner.
[Show abstract][Hide abstract] ABSTRACT: Telomerase-negative immortalized human cells maintain telomeres by alternative lengthening of telomeres (ALT) pathway(s), which may involve homologous recombination. We find that endogenous BLM protein co-localizes with telomeric foci in ALT human cells but not telomerase positive immortal cell lines or primary cells. BLM interacts in vivo with the telomeric protein TRF2 in ALT cells, as detected by FRET and co-immunoprecipitation. Transient over-expression of green fluorescent protein (GFP)-BLM results in marked, ALT cell-specific increases in telomeric DNA. The association of BLM with telomeres and its effect on telomere DNA synthesis require a functional helicase domain. Our results identify BLM as the first protein found to affect telomeric DNA synthesis exclusively in human ALT cells and suggest that BLM facilitates recombination-driven amplification of telomeres in ALT cells.
Preview · Article · Jan 2003 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: Prolonged growth of murine embryonic stem (ES) cells lacking the telomerase reverse transcriptase, mTert, results in a loss of telomere DNA and an increased incidence of end-to-end fusions and aneuploidy. Furthermore, loss of only one copy of mTert also results in telomere shortening intermediate between wild-type (wt) and mTert-null ES cells [Liu, Y., Snow, B. E., Hande, M. P., Yeung, D., Erdmann, N. J., Wakeham, A., Itie, A., Siderovski, D. P., Lansdorp, P. M., Robinson, M. O. & Harrington, L. (2000) Curr. Biol. 10, 1459-1462]. Unexpectedly, although average telomere length in mTert(+/-) ES cells declined to a similar level as mTert-null ES cells, mTert(+/-) ES cell lines retained a minimal telomeric DNA signal at all chromosome ends. Consequently, no end-to-end fusions and genome instability were observed in the latest passages of mTert(+/-) ES cell lines. These data uncover a functional distinction between the dosage-dependent function of telomerase in average telomere-length maintenance and the selective maintenance of critically short telomeres in cells heterozygous for mTert. In normal and tumor cells, we suggest that telomerase activity insufficient to maintain a given average telomere length may, nonetheless, provide a protective advantage from end-to-end fusion and genome instability.
Full-text · Article · Mar 2002 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Electrophoretic separations Microfluidic devices used for electrophoretic separations had simple cross-patterns with 180-µm wide and 35-µm deep channels. Typically, channels were filled by submerging devices in isopropanol (IPA) under vacuum in a dessicator until all gas was removed (~5 min). Then, reservoirs were filled with run buffer which was pushed through the channels with pressure from a pipette. For pinched injections and elecrophoretic separations, fluids were controlled by applying electric fields via a high voltage sequencer (LabSmith, Livermore, CA). Analytes were detected by laser induced fluorescence using an inverted microscope (Olympus IX-71) mated to an argon ion laser (Melles Griot, Carlsbad, CA). The 488-nm laser line was used for green fluorescence (fluorescein and rhodamine), and the 457-nm line was used for blue fluorescence (NDA-derivatives). The laser was focused into the channel using a 60x objective; the fluorescent signal was collected by the same lens and filtered optically (536/40-nm band pass and 488-nm notch filter for green fluorescence and a 482/35-nm band pass and 457-nm notch filter for blue fluorescence) and spatially (500 μm pinhole) and imaged onto a photomultiplier tube (Hamamatsu, Bridgewater, NJ). PMT current was converted to voltage using a picoammeter (Keithley Instruments, Cleveland, OH) which was then collected using a DAQpad A-D converter (National Instruments, Austin, TX) and PC running a custom LabView (Natl. Inst.) program.