Modeling neurodevelopment in a dish with pluripotent stem cells

Pluripotent stem cells (PSCs) can differentiate into all cell types in the body, and their differentiation procedures recapitulate the developmental processes of embryogenesis. Focusing on neurodevelopment, we describe here the application of knowledge gained from embryology to the neural induction of PSCs. Furthermore, PSC‐based neural modeling provides novel insights into neurodevelopmental processes. In particular, human PSC cultures are a powerful tool for the study of human‐specific neurodevelopmental processes and could even enable the elucidation of the mechanisms of human brain evolution. We also discuss challenges and potential future directions in further improving PSC‐based neural modeling.     

Label‐free identification and characterization of human pluripotent stem cell‐derived cardiomyocytes using second harmonic generation (SHG) microscopy

Pluripotent stem cell‐derived cardiomyocytes (PSC‐CMs) are a potentially unlimited source of cardiomyocytes (CMs) for cardiac transplantation therapies. The establishment of pure PSC‐CM populations is important for this application, but is hampered by a lack of CM‐specific surface markers suitable for their identification and sorting. Contemporary purification techniques are either non‐specific or require genetic modification. We report a second harmonic generation (SHG) signal detectable in PSC‐CMs that is attributable to sarcomeric myosin, dependent on PSC‐CM maturity, and retained while PSC‐CMs are in suspension. Our study demonstrates the feasibility of developing a SHG‐activated flow cytometer for the non‐invasive purification of PSC‐CMs. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The specific binding of peptide ligands to cardiomyocytes derived from mouse embryonic stem cells

Purification of pluripotent stem cell (PSC)‐derived cardiomyocytes is critical for the application of cardiomyocytes both in clinical and basic research. Finding a specific cell marker is a promising method for purifying induced cells. The present study employed phage display technology to search for particular cell markers that could bind specifically to PSC‐derived cardiomyocytes. After three rounds of biopanning, several peptides were obtained. The ELISA results show the no. 3 sequence peptide (QPFTTSLTPPAR), and other four sequences having a consensus motif [SS(Q)PPQ(S)], no. 9, 11, 14, and 10, have relatively high affinity and specificity to cardiomyocytes. Immunofluorescence confirmed that the selected peptides could bind specifically to the PSC‐derived cardiomyocytes. Competition tests with chemically synthesized peptides revealed the binding ability was caused by the peptide itself. Western blot analysis proved the phages were both bound to two 17 kDa cardiomyocyte membrane proteins and the no. 9 sequence showed a 55 kDa protein that was not observed in the no. 3 sequence. These results suggest that the selected peptides specifically target receptors on PSC‐derived cardiomyocyte membranes. The results will pave the way for further studies of cell surface markers and their applications, such as labeling, purification, and as vehicles for drug delivery. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Polymerization and matrix physical properties as important design considerations for soluble collagen formulations

Despite extensive use of type I collagen for research and medical applications, its fibril‐forming or polymerization potential has yet to be fully defined and exploited. Here, we describe a type I collagen formulation that is acid solubilized from porcine skin collagen (PSC), quality controlled based upon polymerization potential, and well suited as a platform polymer for preparing three‐dimensional (3D) culture systems and injectable/implantable in vivo cellular microenvironments in which both relevant biochemical and biophysical parameters can be precision‐controlled. PSC is compared with three commercial collagens in terms of composition and purity as well as polymerization potential, which is described by kinetic parameters and fibril microstructure and mechanical properties of formed matrices. When subjected to identical polymerization conditions, PSC showed significantly decreased polymerization times compared to the other collagens and yielded matrices with the greatest mechanical integrity and broadest range of mechanical properties as characterized in oscillatory shear, uniaxial extension, and unconfined compression. Compositional and intrinsic viscosity analyses suggest that the enhanced polymerization potential of PSC may be attributed to its unique oligomer composition. Collectively, this work demonstrates the importance of standardizing next generation collagen formulations based upon polymerization potential and provides preliminary insight into the contribution of oligomers to collagen polymerization properties. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 690–707, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Cryopreservation of pluripotent stem cell aggregates in defined protein‐free formulation

Cultivation of undifferentiated pluripotent stem cells (PSCs) as aggregates has emerged as an efficient culture configuration, enabling rapid and controlled large scale expansion. Aggregate‐based PSC cryopreservation facilitates the integrated process of cell expansion and cryopreservation, but its feasibility has not been demonstrated. The goals of current study are to assess the suitability of cryopreserving intact mouse embryonic stem cell (mESC) aggregates and investigate the effects of aggregate size and the formulation of cryopreservation solution on mESC survival and recovery. The results demonstrated the size‐dependent cell survival and recovery of intact aggregates. In particular, the generation of reactive oxygen species (ROS) and caspase activation were reduced for small aggregates (109 ± 55 μm) compared to medium (245 ± 77 μm) and large (365 ± 141 μm) ones, leading to the improved cell recovery. In addition, a defined protein‐free formulation was tested and found to promote the aggregate survival, eliminating the cell exposure to animal serum. The cryopreserved aggregates also maintained the pluripotent markers and the differentiation capacity into three‐germ layers after thawing. In summary, the cryopreservation of small PSC aggregates in a defined protein‐free formulation was shown to be a suitable approach toward a fully integrated expansion and cryopreservation process at large scale. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Modeling signaling‐dependent pluripotency with Boolean logic to predict cell fate transitions

Pluripotent stem cells (PSCs) exist in multiple stable states, each with specific cellular properties and molecular signatures. The mechanisms that maintain pluripotency, or that cause its destabilization to initiate development, are complex and incompletely understood. We have developed a model to predict stabilized PSC gene regulatory network (GRN) states in response to input signals. Our strategy used random asynchronous Boolean simulations (R‐ABS) to simulate single‐cell fate transitions and strongly connected components (SCCs) strategy to represent population heterogeneity. This framework was applied to a reverse‐engineered and curated core GRN for mouse embryonic stem cells (mESCs) and used to simulate cellular responses to combinations of five signaling pathways. Our simulations predicted experimentally verified cell population compositions and input signal combinations controlling specific cell fate transitions. Extending the model to PSC differentiation, we predicted a combination of signaling activators and inhibitors that efficiently and robustly generated a Cdx2⁺Oct4^? cells from naïve mESCs. Overall, this platform provides new strategies to simulate cell fate transitions and the heterogeneity that typically occurs during development and differentiation.     

Senescence determines the fate of activated rat pancreatic stellate cells

In chronic pancreatitis (CP), persistent activation of pancreatic stellate cells (PSC) converts wound healing into a pathological process resulting in organ fibrosis. Here, we have analysed senescence as a novel mechanism involved in the termination of PSC activation and tissue repair. PSC senescence was first studied in vitro by establishing long‐term cultures and by applying chemical triggers, using senescence‐associated β‐Galactosidase (SA β‐Gal) as a surrogate marker. Subsequently, susceptibility of PSC to immune cell‐mediated cytolysis was investigated employing cocultures. Using the model of dibutyltin dichloride‐induced CP in rats, appearance of senescent cells was monitored by immunohistochemistry and immunofluorescence, and correlated with the progression of tissue damage and repair, immune cell infiltration and fibrosis. The results indicated that long‐term culture and exposure of PSC to stressors (doxorubicin, H₂O₂ and staurosporine) induced senescence. Senescent PSC highly expressed CDKN1A/p21, mdm2 and interleukin (IL)‐6, but displayed low levels of α‐smooth muscle actin. Senescence increased the susceptibility of PSC to cytolysis. In CP, the number of senescent cells correlated with the severity of inflammation and the extension of fibrosis. Areas staining positive for SA β‐Gal overlapped with regions of fibrosis and dense infiltrates of immune cells. Furthermore, a close physical proximity of immune cells and activated PSC was observed. We conclude that inflammation, PSC activation and cellular senescence are timely coupled processes which take place in the same microenvironment of the inflamed pancreas. Lymphocytes may play a dual‐specific role in pancreatic fibrogenesis, triggering both the initiation of wound healing by activating PSC, and its completion by killing senescent stellate cells.     

Serum supplemented culture medium masks hypertrophic phenotypes in human pluripotent stem cell derived cardiomyocytes

It has been known for over 20 years that foetal calf serum can induce hypertrophy in cultured cardiomyocytes but this is rarely considered when examining cardiomyocytes derived from pluripotent stem cells (PSC). Here, we determined how serum affected cardiomyocytes from human embryonic‐ (hESC) and induced pluripotent stem cells (hiPSC) and hiPSC from patients with hypertrophic cardiomyopathy linked to a mutation in the MYBPC3 gene. We first confirmed previously published hypertrophic effects of serum on cultured neonatal rat cardiomyocytes demonstrated as increased cell surface area and beating frequency. We then found that serum increased the cell surface area of hESC‐ and hiPSC‐derived cardiomyocytes and their spontaneous contraction rate. Phenylephrine, which normally induces cardiac hypertrophy, had no additional effects under serum conditions. Likewise, hiPSC‐derived cardiomyocytes from three MYBPC3 patients which had a greater surface area than controls in the absence of serum as predicted by their genotype, did not show this difference in the presence of serum. Serum can thus alter the phenotype of human PSC derived cardiomyocytes under otherwise defined conditions such that the effects of hypertrophic drugs and gene mutations are underestimated. It is therefore pertinent to examine cardiac phenotypes in culture media without or in low concentrations of serum.     

Protein structure comparison by probability-based matching of secondary structure elements

MOTIVATION: Protein structure comparison (PSC) has been used widely in studies of structural and functional genomics. However, PSC is computationally expensive and as a result almost all of the PSC methods currently in use look only for the optimal alignment and ignore many alternative alignments that are statistically significant and that may provide insight into protein evolution or folding. RESULTS: We have developed a new PSC method with efficiency to detect potentially viable alternative alignments in all-against-all database comparisons. The efficiency of the new PSC method derives from the ability to directly home in on a limited number of viable and ranked alignment solutions based on intuitively derived SSE (secondary structure element)-matching probabilities.     

Bringing Stem Cell‐Based Therapies for Type 1 Diabetes to the Clinic: Early Insights from Bioprocess Economics and Cost‐Effectiveness Analysis

Differentiation of pluripotent stem cells (PSCs) into β cells could provide insulin independence for type 1 diabetes (T1D) patients. This approach would reduce the clinical complications that most patients managed on intensive insulin therapy (IIT) face. However, bottlenecks of PSC manufacturing and limited engraftment of encapsulated cells hinder the long‐term effectiveness of these therapies. A bioprocess decision‐support tool is combined with a disease state‐transition model to evaluate the cost‐effectiveness of the stem cell‐based therapy against IIT. Clinical effectiveness is assessed in quality‐adjusted life years (QALYs). Manufacturing costs per patient reduce from $430 000 to $160 000 with optimization of batch size and annual demand. For 96% of the patients, cell therapy improves the quality of life compared to IIT. Cost savings are achieved for 2% of the population through prevention of renal disease. The therapy is cost‐effective for 3.4% of patients when a willingness to pay (WTP) of up to $150 000 per QALY is considered. A 75% cost reduction in the cell therapy price increases cost‐effectiveness likelihood to 51% at $100 000 per QALY. This study highlights the need for scalable manufacturing platforms for stem cell therapies, as well as to prioritizing access to the therapy to patients with an increased likelihood of costly complications.     

Spectral Engineering of Semitransparent Polymer Solar Cells for Greenhouse Applications

In this study, a wavelength selective semitransparent polymer solar cell (ST‐PSC) with a proper transmission spectrum for plant growth is proposed for greenhouse applications. A ternary strategy combining a wide bandgap polymer donor with a near‐infrared absorbing nonfullerene acceptor and a high electron mobility fullerene acceptor is introduced to achieve PSCs with power conversion efficiency (PCE) over 10%. The addition of PC₇₁BM into J52:IEICO‐4F binary blend contributes to the suppressed trap‐assisted recombination, enhanced charge extraction, and improved open‐circuit voltage simultaneously. ST‐PSC based on the J52:IEICO‐4F:PC₇₁BM ternary blend shows an optimized performance with PCE of 7.75% and a defined crop growth factor of 24.8%. Such high‐performance ST‐PSC is achieved by carefully engineering the absorption spectrum of the light harvesting materials. As a result, the transmission spectra of the semitransparent devices are well‐matched with the absorption spectra of the photoreceptors, such as chlorophylls, in green plants, which provides adequate lighting conditions for photosynthesis and plant growth, and therefore making it a competitive candidate for photovoltaic greenhouse applications.     

Progress in Tandem Solar Cells Based on Hybrid Organic–Inorganic Perovskites

Owing to their high efficiency, low‐cost solution‐processability, and tunable bandgap, perovskite solar cells (PSCs) made of hybrid organic‐inorganic perovskite (HOIP) thin films are promising top‐cell candidates for integration with bottom‐cells based on Si or other low‐bandgap solar‐cell materials to boost the power conversion efficiency (PCE) beyond the Shockley‐Quiesser (S‐Q) limit. In this review, recent progress in such tandem solar cells based on the emerging PSCs is summarized and reviewed critically. Notable achievements for different tandem solar cell configurations including mechanically‐stacked, optical coupling, and monolithically‐integrated with PSCs as top‐cells are described in detail. Highly‐efficient semitransparent PSC top‐cells with high transmittance in near‐infrared (NIR) region are critical for tandem solar cells. Different types of transparent electrodes with high transmittance and low sheet‐resistance for PSCs are reviewed, which presents a grand challenge for PSCs. The strategies to obtain wide‐bandgap PSCs with good photo‐stability are discussed. The PCE reduction due to reflection loss, parasitic absorption, electrical loss, and current mismatch are analyzed to provide better understanding of the performance of PSC‐based tandem solar cells.     

Engineering stem cell fate with biochemical and biomechanical properties of microcarriers

Microcarriers have been widely used for various biotechnology applications because of their high scale‐up potential, high reproducibility in regulating cellular behavior, and well‐documented compliance with current Good Manufacturing Practices (cGMP). Recently, microcarriers have been emerging as a novel approach for stem cell expansion and differentiation, enabling potential scale‐up of stem cell‐derived products in large bioreactors. This review summarizes recent advances of using microcarriers in mesenchymal stem cell (MSC) and pluripotent stem cell (PSC) cultures. From the reported data, efficient expansion and differentiation of stem cells on microcarriers rely on their ability to modulate cell shape (i.e. round or spreading) and cell organization (i.e. aggregate size). Nonetheless, current screening of microcarriers remains empirical, and accurate understanding of how stem cells interact with microcarriers still remains unknown. This review suggests that accurate characterization of biochemical and biomechanical properties of microcarriers is required to fully exploit their potential in regulating stem cell fate decision. Due to the variety of microcarriers, such detailed analyses should lead to the rational design of application‐specific microcarriers, enabling the exploitation of reproducible effects for large scale biomedical applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1354–1366, 2013     

Three‐dimensional neural differentiation of embryonic stem cells with ACM induction in microfibrous matrices in bioreactors

The clinical use of pluripotent stem cell (PSC)‐derived neural cells requires an efficient differentiation process for mass production in a bioreactor. Toward this goal, neural differentiation of murine embryonic stem cells (ESCs) in three‐dimensional (3D) polyethylene terephthalate microfibrous matrices was investigated in this study. To streamline the process and provide a platform for process integration, the neural differentiation of ESCs was induced with astrocyte‐conditioned medium without the formation of embryoid bodies, starting from undifferentiated ESC aggregates expanded in a suspension bioreactor. The 3D neural differentiation was able to generate a complex neural network in the matrices. When compared to 2D differentiation, 3D differentiation in microfibrous matrices resulted in a higher percentage of nestin‐positive cells (68% vs. 54%) and upregulated gene expressions of nestin, Nurr1, and tyrosine hydroxylase. High purity of neural differentiation in 3D microfibrous matrix was also demonstrated in a spinner bioreactor with 74% nestin + cells. This study demonstrated the feasibility of a scalable process based on 3D differentiation in microfibrous matrices for the production of ESC‐derived neural cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1013–1022, 2013     

Characterization of 3D pluripotent stem cell aggregates and the impact of their properties on bioprocessing

Pluripotent stem cells (PSCs) have been traditionally expanded on a two-dimensional (2D) surface and require substrates coated with extracellular matrix (ECM) proteins. Recently, PSCs have been successfully expanded in suspension as undifferentiated PSC aggregates, which offer a means for large-scale production. Toward lineage-specific differentiation, PSCs can form aggregate-like structures known as embryoid bodies (EBs). The morphology and size of EBs have been shown to significantly affect the differentiation into specific lineages and three-dimensional (3D) tissue development, thus efforts have been devoted to form size-controlled EBs. The integration of both PSC expansion and differentiation in suspension promotes PSC-derived cell production in bioreactors. However, the cellular organization and differentiation potential of PSC aggregates, as well as the role of the cues provided by the reactors to regulate EB fate, have yet to be fully understood. Despite these challenges, integrated PSC aggregate-based culture provides a platform for a simple, scalable bioprocess for the potential application of PSCs in regenerative medicine, disease modeling, and drug discovery.     

Control of Mesoscale Morphology and Photovoltaic Performance in Diketopyrrolopyrrole‐Based Small Band Gap Terpolymers

Morphology control is one of the key strategies in optimizing the performance of organic photovoltaic materials, particularly for diketopyrrolopyrrole (DPP)‐based donor polymers. The design of DPP‐based polymers that provide high power conversion efficiency (PCE) presents a significant challenge that requires optimization of both energetics and morphology. Herein, a series of high performance, small band gap DPP‐based terpolymers are designed via two‐step side chain engineering, namely introducing alternating short and long alkyls for reducing the domain spacing and inserting alkylthio for modulating the energy levels. The new DPP‐based terpolymers are compared to delineate how the side chain impacts the mesoscale morphology. By employing the alkylthio‐substituted terpolymer PBDPP‐TS, the new polymer solar cell (PSC) device realizes a good balance of a high V _oc of 0.77 V and a high J _sc over 15 mA cm^?2, and thus realizes desirable PCE in excess of 8% and 9.5% in single junction and tandem PSC devices, respectively. The study indicates better control of domain purity will greatly improve performance of single junction DPP‐based PSCs toward 10% efficiency. More significantly, the utility of this stepwise side chain engineering can be readily expanded to other classes of well‐defined copolymers and triggers efficiency breakthroughs in novel terpolymers for photovoltaic and related electronic applications.     

hiPSC‐derived iMSCs: NextGen MSCs as an advanced therapeutically active cell resource for regenerative medicine

Mesenchymal stem cells (MSCs) are being assessed for ameliorating the severity of graft‐versus‐host disease, autoimmune conditions, musculoskeletal injuries and cardiovascular diseases. While most of these clinical therapeutic applications require substantial cell quantities, the number of MSCs that can be obtained initially from a single donor remains limited. The utility of MSCs derived from human‐induced pluripotent stem cells (hiPSCs) has been shown in recent pre‐clinical studies. Since adult MSCs have limited capability regarding proliferation, the quantum of bioactive factor secretion and immunomodulation ability may be constrained. Hence, the alternate source of MSCs is being considered to replace the commonly used adult tissue‐derived MSCs. The MSCs have been obtained from various adult and foetal tissues. The hiPSC‐derived MSCs (iMSCs) are transpiring as an attractive source of MSCs because during reprogramming process, cells undergo rejuvination, exhibiting better cellular vitality such as survival, proliferation and differentiations potentials. The autologous iMSCs could be considered as an inexhaustible source of MSCs that could be used to meet the unmet clinical needs. Human‐induced PSC‐derived MSCs are reported to be superior when compared to the adult MSCs regarding cell proliferation, immunomodulation, cytokines profiles, microenvironment modulating exosomes and bioactive paracrine factors secretion. Strategies such as derivation and propagation of iMSCs in chemically defined culture conditions and use of footprint‐free safer reprogramming strategies have contributed towards the development of clinically relevant cell types. In this review, the role of iPSC‐derived mesenchymal stromal cells (iMSCs) as an alternate source of therapeutically active MSCs has been described. Additionally, we also describe the role of iMSCs in regenerative medical applications, the necessary strategies, and the regulatory policies that have to be enforced to render iMSC's effectiveness in translational medicine.     

Fully Solution‐Processed TCO‐Free Semitransparent Perovskite Solar Cells for Tandem and Flexible Applications

Semitransparent perovskite solar cells (st‐PSCs) have received remarkable interest in recent years because of their great potential in applications for solar window, tandem solar cells, and flexible photovoltaics. However, all reported st‐PSCs require expensive transparent conducting oxides (TCOs) or metal‐based thin films made by vacuum deposition, which is not cost effective for large‐scale fabrication: the cost of TCOs is estimated to occupy ≈75% of the manufacturing cost of PSCs. To address this critical challenge, this study reports a low‐temperature and vacuum‐free strategy for the fabrication of highly efficient TCO‐free st‐PSCs. The TCO‐free st‐PSC on glass exhibits 13.9% power conversion efficiency (PCE), and the four‐terminal tandem cell made with the st‐PSC top cell and c‐Si bottom cell shows an overall PCE of 19.2%. Due to the low processing temperature, the fabrication of flexible st‐PSCs is demonstrated on polyethylene terephthalate and polyimide, which show excellent stability under repeated bending or even crumbing.