Peter W. Zandstra

Growth factors, matrices, and forces combine and control stem cells

Stem cell fate is influenced by a number of factors and interactions that require robust control for safe and effective regeneration of functional tissue. Coordinated interactions with soluble factors, other cells, and extracellular matrices define a local biochemical and mechanical niche with complex and dynamic regulation that stem cells sense. Decellularized tissue matrices and synthetic polymer niches are being used in the clinic, and they are also beginning to clarify fundamental aspects of how stem cells contribute to homeostasis and repair, for example, at sites of fibrosis. Multifaceted technologies are increasingly required to produce and interrogate cells ex vivo, to build predictive models, and, ultimately, to enhance stem cell integration in vivo for therapeutic benefit.

Reproducible, Ultra High-Throughput Formation of Multicellular Organization from Single Cell Suspension-Derived Human Embryonic Stem Cell Aggregates

Background 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. Methodology/Principal Findings 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 cm2. Conclusions/Significance 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.

Shear‐Controlled Single‐Step Mouse Embryonic Stem Cell Expansion and Embryoid Body–Based Differentiation

To facilitate the exploitation of embryonic stem cells (ESCs) and ESC‐derived cells, scale‐up of cell production and optimization of culture conditions are necessary. Conventional ESC culture methods are impractical for large‐scale cell production and lack robust microenvironmental control. We developed two stirred‐suspension culture systems for the propagation of undifferentiated ESCs—microcarrier and aggregate cultures—and compared them with tissue‐culture flask and Petri dish controls. ESCs cultured on glass microcarriers had population doubling times (∼14–17 hours) comparable to tissue‐culture flask controls. ESC growth could be elicited in shear‐controlled stirred‐suspension culture, with population doubling times ranging between 24 and 39 hours at 100 rpm impeller speed. Upon removal of leukemia inhibitory factor, the size‐controlled ESC aggregates developed into embryoid bodies (EBs) capable of multilineage differentiation. A comprehensive analysis of ESC developmental potential, including flow cytometry for Oct‐4, SSEA‐1, and E‐cadherinprotein expression, reverse transcription–polymerase chain reaction for Flk‐1, HNF3‐β, MHC, and Sox‐1 gene expression, and EB differentiation analysis, demonstrated that the suspension‐cultured ESCs retained the developmental potential of the starting cell population. Analysis of E‐cadherin−/− and E‐cadherin+/− cells using both systems provided insight into the mechanisms behind the role of cell aggregation control, which is fundamental to these observations. These cell‐culture tools should prove useful for both the production of ESCs and ESC‐derived cells and for investigations into adhesion, survival, and differentiation phenomena during ESC propagation and differentiation.

The Systematic Production of Cells for Cell Therapies

Stem cells have emerged as the starting material of choice for bioprocesses to produce cells and tissues to treat degenerative, genetic, and immunological disease. Translating the biological properties and potential of stem cells into therapies will require overcoming significant cell-manufacturing and regulatory challenges. Bioprocess engineering fundamentals, including bioreactor design and process control, need to be combined with cellular systems biology principles to guide the development of next-generation technologies capable of producing cell-based products in a safe, robust, and cost-effective manner. The step-wise implementation of these bioengineering strategies will enhance cell therapy product quality and safety, expediting clinical development.

Generation of human embryonic stem cell‐derived mesoderm and cardiac cells using size‐specified aggregates in an oxygen‐controlled bioreactor

The ability to generate human pluripotent stem cell‐derived cell types at sufficiently high numbers and in a reproducible manner is fundamental for clinical and biopharmaceutical applications. Current experimental methods for the differentiation of pluripotent cells such as human embryonic stem cells (hESC) rely on the generation of heterogeneous aggregates of cells, also called “embryoid bodies” (EBs), in small scale static culture. These protocols are typically (1) not scalable, (2) result in a wide range of EB sizes and (3) expose cells to fluctuations in physicochemical parameters. With the goal of establishing a robust bioprocess we first screened different scalable suspension systems for their ability to support the growth and differentiation of hESCs. Next homogeneity of initial cell aggregates was improved by employing a micro‐printing strategy to generate large numbers of size‐specified hESC aggregates. Finally, these technologies were integrated into a fully controlled bioreactor system and the impact of oxygen concentration was investigated. Our results demonstrate the beneficial effects of stirred bioreactor culture, aggregate size‐control and hypoxia (4% oxygen tension) on both cell growth and cell differentiation towards cardiomyocytes. QRT‐PCR data for markers such as Brachyury, LIM domain homeobox gene Isl‐1, Troponin T and Myosin Light Chain 2v, as well as immunohistochemistry and functional analysis by response to chronotropic agents, documented the impact of these parameters on cardiac differentiation. This study provides an important foundation towards the robust generation of clinically relevant numbers of hESC derived cells. Biotechnol. Bioeng. 2009;102: 493–507.

An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming.

Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. Here, we identify an evolutionarily conserved embryonic stem cell (ESC)-specific AS event that changes the DNA-binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency, including OCT4, NANOG, NR5A2, and GDF3, while concomitantly repressing genes required for ESC differentiation. This isoform also promotes the maintenance of ESC pluripotency and contributes to efficient reprogramming of somatic cells into induced pluripotent stem cells. These results reveal a pivotal role for an AS event in the regulation of pluripotency through the control of critical ESC-specific transcriptional programs.

Functional immobilization of signaling proteins enables control of stem cell fate

The mode of ligand presentation has a fundamental role in organizing cell fate throughout development. We report a rapid and simple approach for immobilizing signaling ligands to maleic anhydride copolymer thin-film coatings, enabling stable signaling ligand presentation at interfaces at defined concentrations. We demonstrate the utility of this platform technology using leukemia inhibitory factor (LIF) and stem cell factor (SCF). Immobilized LIF supported mouse embryonic stem cell (mESC) pluripotency for at least 2 weeks in the absence of added diffusible LIF. Immobilized LIF activated signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) signaling in a dose-dependent manner. The introduced method allows for the robust investigation of cell fate responses from interface-immobilized ligands.

Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal

Human adult stem cell expansion Transfused blood saves lives. Despite the widespread use of this critical resource, it is difficult to increase blood cell numbers outside of the body. By screening thousands of small compounds, Fares et al. identify a molecule that expands human stem cell numbers in cord blood. The researchers generate many variations of that molecule and show that one such compound provides even greater human blood cell expansion. If researchers can provide increased numbers of stem cells and progenitor cells, cord blood should find even greater use in the clinic. Science, this issue p. 1509 The self-renewal of human hematopoietic stem cells in vitrois enhanced by the pyrimidoindole derivative UM171. The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy.

Rapid Expansion of Human Hematopoietic Stem Cells by Automated Control of Inhibitory Feedback Signaling

Clinical hematopoietic transplantation outcomes are strongly correlated with the numbers of cells infused. Anticipated novel therapeutic implementations of hematopoietic stem cells (HSCs) and their derivatives further increase interest in strategies to expand HSCs ex vivo. A fundamental limitation in all HSC-driven culture systems is the rapid generation of differentiating cells and their secreted inhibitory feedback signals. Herein we describe an integrated computational and experimental strategy that enables a tunable reduction in the global levels and impact of paracrine signaling factors in an automated closed-system process by employing a controlled fed-batch media dilution approach. Application of this system to human cord blood cells yielded a rapid (12-day) 11-fold increase of HSCs with self-renewing, multilineage repopulating ability. These results highlight the marked improvements that control of feedback signaling can offer primary stem cell culture and demonstrate a clinically relevant rapid and relatively low culture volume strategy for ex vivo HSC expansion.

Design and formulation of functional pluripotent stem cell-derived cardiac microtissues

Significance Robust and predictive in vitro models of human cardiac tissue function could have transformative impact on our ability to test new drugs and understand cardiac disease. Despite significant effort, the generation of high-fidelity adult-like human cardiac tissue analogs remains challenging. In this paper, we systematically explore the design criteria for pluripotent stem cell-derived engineered cardiac tissue. Parameters such as biomechanical stress during tissue remodeling, input-cell composition, electrical stimulation, and tissue geometry are evaluated. Our results suggest that a specified combination of a 3D matrix-based microenvironment, uniaxial mechanical stress, and mixtures of cardiomyocytes and fibroblasts improves the performance and maturation state of in vitro engineered cardiac tissue. Access to robust and information-rich human cardiac tissue models would accelerate drug-based strategies for treating heart disease. Despite significant effort, the generation of high-fidelity adult-like human cardiac tissue analogs remains challenging. We used computational modeling of tissue contraction and assembly mechanics in conjunction with microfabricated constraints to guide the design of aligned and functional 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues that we term cardiac microwires (CMWs). Miniaturization of the platform circumvented the need for tissue vascularization and enabled higher-throughput image-based analysis of CMW drug responsiveness. CMW tissue properties could be tuned using electromechanical stimuli and cell composition. Specifically, controlling self-assembly of 3D tissues in aligned collagen, and pacing with point stimulation electrodes, were found to promote cardiac maturation-associated gene expression and in vivo-like electrical signal propagation. Furthermore, screening a range of hPSC-derived cardiac cell ratios identified that 75% NKX2 Homeobox 5 (NKX2-5)+ cardiomyocytes and 25% Cluster of Differentiation 90 OR (CD90)+ nonmyocytes optimized tissue remodeling dynamics and yielded enhanced structural and functional properties. Finally, we demonstrate the utility of the optimized platform in a tachycardic model of arrhythmogenesis, an aspect of cardiac electrophysiology not previously recapitulated in 3D in vitro hPSC-derived cardiac microtissue models. The design criteria identified with our CMW platform should accelerate the development of predictive in vitro assays of human heart tissue function.