Optimal Cell Survival Research Articles

Recent developments in cell shipping methods.

As opposed to remarkable advances in the cell therapy industry, research reveal inexplicable difficulties associated with preserving and post-thawing cell death. Post cryopreservation apoptosis is a common occurrence that has attracted the attention of scientists to use apoptosis inhibitors. Transporting cells without compromising their survival and function is crucial for any experimental cell-based therapy. Preservation of cells allows the safe transportation of cells between distances and improves quality control testing in clinical and research applications. The vitality of transported cells is used to evaluate the efficacy of transportation strategies. For many decades, the conventional global methods of cell transfer were not only expensive but also challenging and had adverse effects. The first determination of some projects is optimizing cell survival after cryopreservation. The new generation of cryopreservation science wishes to find appropriate and alternative methods for cell transportation to ship viable cells at an ambient temperature without dry ice or in media-filled flasks. The diversity of cell therapies demands new cell shipping methodologies and cryoprotectants. In this review, we tried to summarize novel improved cryopreservation methods and alternatives to cryopreservation with safe and viable cell shipping at ambient temperature, including dry preservation, hypothermic preservation, gel-based methods, encapsulation methods, fibrin microbeads, and osmolyte solution compositions.

P-150 clinical pregnancy rates of oocyte donor cycles are similar when universal media and/or oocyte-specific media are used for oocyte vitrification and warming

Abstract Study question Did the use of universal vitrification/warming media have an impact on the clinical pregnancy rates in oocyte donation cycles compared with oocyte-specific vitrification/warming media? Summary answer Using universal media for oocyte vitrification and warming yields similar clinical pregnancy rates but higher oocyte survival rates than oocyte-specific media. What is known already Vitrification is now the gold standard for oocyte and embryo cryopreservation. Several commercial kits are available, some are designed for specific cell types (e.g., oocytes, zygotes, cleavage-stage embryos or blastocysts) and others are suitable for several stages, therefore termed “universal”. While the composition and the exposition protocol of stage specific media are optimized for specific cell types, “universal” media display a single composition, therefore exposition protocols need to be adapted to each specific developmental stage to ensure optimal cell survival rates. The potential use of the universal media allows to optimize the management of media in ART laboratories. Study design, size, duration This is a retrospective monocentric study. We compared the results obtained in our center for oocyte vitrification in donor cycles with the successive use of oocyte-specific vitrification/warming media (RapidVit/Warm™ Oocyte, Vitrolife) and afterwards universal vitrification/warming media (RapidVit/Warm™ Omni, Vitrolife). We analyzed 111 oocyte recipient ICSI cycles performed between March 2016 and July 2020 (86 recipient couples) and their 81 corresponding oocyte donation cycles (79 donors) with these media. Participants/materials, setting, methods Oocyte recipient ICSI cycles were divided in three groups depending on the combination of vitrification and warming media used for the oocyte vitrification and warming procedures: “specific/specific” (S/S), “specific/universal” (S/U) and “universal/universal” (U/U). The primary outcome was the clinical pregnancy rate per embryo transfer. Secondary outcomes were the oocyte survival rates, fertilization rates, cleavage and blastocyst rates, live birth and miscarriage rates. Main results and the role of chance The age of the donors (31.7+/-3.6 yo) and the recipients (35.1+/-4.5) was similar for the three groups (p > 0.05). Total FSH dose was similar for the 3 groups (1814.2+/-505UI) (p > 0.05) Clinical pregnancy rates were similar when universal vitrification/warming media were used (25.6% for S/U; 25.8% for U/U) compared with oocyte-specific vitrification/warming media (12.2% for S/S, p = 0.25). We observed higher oocyte survival rates when universal media were used (93.7% for U/U, 85.5% for S/U) compared with the use of the oocyte-specific media (75.6% for S/S, p < 0.0001). Fertilization (74.9% for U/U; 68% for S/U; 68.1% for S/S), cleavage (97.9% for U/U; 94.6% for S/U; 89.3% for S/S), and blastulation rates (46.6% for U/U; 50.3% for S/U; 41.7% for S/S) were not different between the three groups (p > 0.05). There was no difference in miscarriage (6.5% for U/U; 10.2% for S/U; 4.9% for S/S) and live birth rates (19.4% for U/U; 15.4% for S/U; 7.3% for S/S) between the 3 groups (p > 0.05). Limitations, reasons for caution The preliminary results of this retrospective study need to be confirmed by a larger prospective study. Wider implications of the findings The use of vitrification/warming universal media, which improves the management of media used for vitrification of oocytes and embryos in ART laboratory, allows to obtain similar clinical pregnancy rates for oocyte recipient donors. Trial registration number not applicable

Commercial and Instant Coffees Effectively Lower Aβ1-40 and Aβ1-42 in N2a/APPswe Cells.

BackgroundAlzheimer's disease (AD) is a multifactorial neurological disease with neurofibrillary tangles and neuritic plaques as histopathological markers. Due to this, although AD is the leading cause of dementia worldwide, clinical AD dementia cannot be certainly diagnosed until neuropathological post-mortem evaluation. Coffee has been reported to have neurologically protective factors, particularly against AD, but coffee brand and type have not been taken into consideration in previous studies. We examined the discrepancies among popular commercial and instant coffees in limiting the development and progression through Aβ1-40 and Aβ1-42 production, and hypothesized that coffee consumption, regardless of brand or type, is beneficial for stalling the progression and development of Aβ-related AD.MethodsCoffee samples from four commercial coffee brands and four instant coffees were purchased or prepared following given instructions and filtered for the study. 5, 2.5, and 1.25% concentrations of each coffee were used to treat N2a/APPswe cell lines. MTT assay was used to assess cell viability for coffee concentrations, as well as pure caffeine concentrations. Sandwich ELISA assay was used to determine Aβ concentration for Aβ1-40 and Aβ1-42 peptides of coffee-treated cells.ResultsCaffeine concentrations were significantly varied among all coffees (DC vs. MDC, PC, SB, NIN, MIN p < 0.05). There was no correlation between caffeine concentration and cell toxicity among brands and types of coffee, with no toxicity at 0.5 mg/ml caffeine and lower. Most coffees were toxic to N2a/APPswe cells at 5% (p < 0.05), but not at 2.5%. Most coffees at a 2.5% concentration reduced Aβ1-40 and Aβ1-42 production, with comparable results between commercial and instant coffees.ConclusionAll coffees tested have beneficial health effects for AD through lowering Aβ1-40 and Aβ1-42 production, with Dunkin' Donuts® medium roast coffee demonstrating the most consistent and optimal cell survival rates and Aβ concentration. On the other hand, Starbucks® coffee exhibited the highest cell toxicity rates among the tested coffees.

Activation-dependent post-transcriptional regulation of CD40L mediates B cell development and survival in germinal centers

Abstract We have described a posttranscriptional pathway of induced mRNA stability that occurs at late times of T cell activation and is engaged by the binding of a polypyrimidine tract-binding protein (PTBP1) complex to the 3′UTR of the CD40L transcript. We explored the in vivo role of this pathway by generating a mouse bearing a deletion of the CD40L stability element (termed CD40LΔ5) and found that Tfh cells harboring the stability defect expressed approximately 60% of WT CD40L. Importantly, this decrease in CD40L corresponded to a poorly elaborated germinal center (GC) response which included significantly decreased levels of switched antibodies, antibody-secreting cells, GL7+ B cells and differentiated GC B cell subsets. In contrast, there was little difference in either somatic hypermutation or affinity maturation between B cells from WT compared to CD40LD5 mice. Notably, both the number and percentage of early memory B cells and plasmablasts were significantly decreased indicating that CD40L expression linked to mRNA stability was critical for B cell differentiation. To understand the impact of this stability pathway on gene expression, CD19+ B cells from CD40LD5 immunized mice were transcriptionally profiled using RNAseq and pathways associated with cell survival and proliferation were downregulated and apoptotic pathways upregulated compared to WT B cells. Importantly, the level of AID mRNA was unchanged whereas c-myc expression was significantly reduced in GL7+ B cells. Together these findings reveal that the CD40L stability element plays a critical role in regulating CD40L expression in Tfh cells, which in turn supports the optimal proliferation and cell survival of high affinity GC B cells and the differentiation of B cell subsets.

Preclinical Efficacy and Safety of a Human Embryonic Stem Cell-Derived Midbrain Dopamine Progenitor Product, MSK-DA01.

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra leading to disabling deficits. Dopamine neuron grafts may provide a significant therapeutic advance over current therapies. We have generated midbrain dopamine neurons from human embryonic stem cells and manufactured large-scale cryopreserved dopamine progenitors for clinical use. After optimizing cell survival and phenotypes in short-term studies, the cell product, MSK-DA01, was subjected to an extensive set of biodistribution, toxicity, and tumorigenicity assessments in mice under GLP conditions. A large-scale efficacy study was also performed in rats with the same lot of cells intended for potential human use and demonstrated survival of the grafted cells and behavioral amelioration in 6-hydroxydopamine lesioned rats. There were no adverse effects attributable to the grafted cells, no obvious distribution outside the brain, and no cell overgrowth or tumor formation, thus paving the way for a future clinical trial.

Natural deep eutectic systems for nature‐inspired cryopreservation of cells

AbstractNatural deep eutectic systems (NADES) are emerging as potential cryoprotective agents (CPA) for cell preservation. In this investigation, we develop an optimized CPA formulation using trehalose‐glycerol NADES (T:G) diluted in Normosol‐R and supplemented with isoleucine. Differential scanning calorimetry (DSC) is used to define the thermophysical properties of NADES‐based solutions, and Raman spectroscopy is used to characterize the effect of NADES on ice formation and hydrogen bonding. Jurkat cells are cryopreserved in each solution, and post‐thaw cell recovery, apoptosis, and growth are quantified. Raman spectra and heat maps show that NADES suppresses both ice formation and dehydration of the nonfrozen region. Supplementing NADES with isoleucine does not affect the solution's thermophysical properties but significantly improves the cells' survival and proliferation post‐thaw. The study indicates that thermophysical properties of CPA solutions alone cannot predict optimal cell survival, suggesting that stabilization of biological structures by CPAs may play a role in successful cryopreservation.

Proteomic analysis of protein homeostasis and aggregation

Protein homeostasis (proteostasis) refers to the ability of cells to preserve the correct balance between protein synthesis, folding and degradation. Proteostasis is essential for optimal cell growth and survival under stressful conditions. Various extracellular and intracellular stresses including heat shock, oxidative stress, proteasome malfunction, mutations and aging-related modifications can result in disturbed proteostasis manifested by enhanced misfolding and aggregation of proteins. To limit protein misfolding and aggregation cells have evolved various strategies including molecular chaperones, proteasome system and autophagy. Molecular chaperones assist folding of proteins, protect them from denaturation and facilitate renaturation of the misfolded polypeptides, whereas proteasomes and autophagosomes remove the irreversibly damaged proteins. The impairment of proteostasis results in protein aggregation that is a major pathological hallmark of numerous age-related disorders, such as cataract, Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases. To discover protein markers and speed up diagnosis of neurodegenerative diseases accompanied by protein aggregation, proteomic tools have increasingly been used in recent years. Systematic and exhaustive analysis of the changes that occur in the proteomes of affected tissues and biofluids in humans or in model organisms is one of the most promising approaches to reveal mechanisms underlying protein aggregation diseases, improve their diagnosis and develop therapeutic strategies.Significance: In this review we outline the elements responsible for maintaining cellular proteostasis and present the overview of proteomic studies focused on protein-aggregation diseases. These studies provide insights into the mechanisms responsible for age–related disorders and reveal new potential biomarkers for Alzheimer’s, Parkinson’s, Huntigton’s and prion diseases.

Activation of neutral sphingomyelinase 2 by starvation induces cell-protective autophagy via an increase in Golgi-localized ceramide

Autophagy is essential for optimal cell function and survival, and the entire process accompanies membrane dynamics. Ceramides are produced by different enzymes at different cellular membrane sites and mediate differential signaling. However, it remains unclear which ceramide-producing pathways/enzymes participate in autophagy regulation under physiological conditions such as nutrient starvation, and what the underlying mechanisms are. In this study, we demonstrate that among ceramide-producing enzymes, neutral sphingomyelinase 2 (nSMase2) plays a key role in autophagy during nutrient starvation. nSMase2 was rapidly and stably activated upon starvation, and the enzymatic reaction in the Golgi apparatus facilitated autophagy through the activation of p38 MAPK and inhibition of mTOR. Moreover, nSMase2 played a protective role against cellular damage depending on autophagy. These findings suggest that nSMase2 is a novel regulator of autophagy and provide evidence that Golgi-localized ceramides participate in cytoprotective autophagy against starvation.

The plasma membrane calcium pumps-The old and the new.

The plasma membrane Ca2+-ATPase (PMCA) pumps play a critical role in the maintenance of calcium (Ca2+) homeostasis, crucial for optimal neuronal function and cell survival. Loss of Ca2+ homeostasis is a key precursor in neuronal dysfunction associated with brain aging and in the pathogenesis of neurodegenerative disorders. In this article, we review evidence showing age-related changes in the PMCAs in synaptic plasma membranes (SPMs) and lipid raft microdomains isolated from rat brain. Both PMCA activity and protein levels decline progressively with increasing age. However, the loss of activity is disproportionate to the reduction of protein levels suggesting the presence of dysfunctional PMCA molecules in aged brain. PMCA activity is also diminished in post-mortem human brain samples from Alzheimer's disease and Parkinson's disease patients and in cell models of these neurodegenerative disorders. Experimental reduction of the PMCAs not only alter Ca2+ homeostasis but also have diverse effects on neurons such as reduced neuritic network, impaired release of neurotransmitter and increased susceptibility to stressful stimuli, particularly to agents that elevate intracellular Ca2+ [Ca2+]i. Loss of PMCA is likely to contribute to neuronal dysfunction observed in the aging brain and in the development of age-dependent neurodegenerative disorders. Therapeutic (pharmacological and/or non-pharmacological) approaches that can enhance PMCA activity and stabilize [Ca2+]i homeostasis may be capable of preventing, slowing, and/or reversing neuronal degeneration.

Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach.

Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i-AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4’s in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we applied substrate trapping coupled with mass spectrometry-based peptide identification in order to extend the list of FTSH4’s physiological substrates and interaction partners. Our analyses revealed, among several putative targets of FTSH4, novel (mitochondrial pyruvate carrier 4 (MPC4) and Pam18-2) and known (Tim17-2) substrates of this protease. Furthermore, we demonstrate that FTSH4 degrades oxidatively damaged proteins in mitochondria. Our report provides new insights into the function of FTSH4 in the maintenance of plant mitochondrial proteome.

Cellular hormetic response to 27-hydroxycholesterol promotes neuroprotection through AICD induction of MAST4 abundance and kinase activity

The function of the amyloid precursor protein (APP) in brain health remains unclear. This study elucidated a novel cytoprotective signaling pathway initiated by the APP transcriptionally active intracellular domain (AICD) in response to 27-hydroxycholesterol (27OHC), an oxidized cholesterol metabolite associated with neurodegeneration. The cellular response to 27OHC was hormetic, such that low, but not high, doses promoted AICD transactivation of microtubule associated serine/threonine kinase family member 4 (MAST4). MAST4 in turn phosphorylated and inhibited FOXO1-dependent transcriptional repression of rhotekin 2 (RTKN2), an oxysterol stress responder, to optimize cell survival. A palmitate-rich diet, which increases serum 27OHC, or APP ablation, abrogated this response in vivo. Further, this pathway was downregulated in human Alzheimer’s Disease (AD) brains but not in frontotemporal dementia brains. These results unveil MAST4 as functional kinase of FOXO1 in a 27OHC AICD-driven, hormetic pathway providing insight for therapeutic approaches against cholesterol associated neuronal disorders.

Gene duplication and co-evolution of G1/S transcription factor specificity in fungi are essential for optimizing cell fitness.

Transcriptional regulatory networks play a central role in optimizing cell survival. How DNA binding domains and cis-regulatory DNA binding sequences have co-evolved to allow the expansion of transcriptional networks and how this contributes to cellular fitness remains unclear. Here we experimentally explore how the complex G1/S transcriptional network evolved in the budding yeast Saccharomyces cerevisiae by examining different chimeric transcription factor (TF) complexes. Over 200 G1/S genes are regulated by either one of the two TF complexes, SBF and MBF, which bind to specific DNA binding sequences, SCB and MCB, respectively. The difference in size and complexity of the G1/S transcriptional network across yeast species makes it well suited to investigate how TF paralogs (SBF and MBF) and DNA binding sequences (SCB and MCB) co-evolved after gene duplication to rewire and expand the network of G1/S target genes. Our data suggests that whilst SBF is the likely ancestral regulatory complex, the ancestral DNA binding element is more MCB-like. G1/S network expansion took place by both cis- and trans- co-evolutionary changes in closely related but distinct regulatory sequences. Replacement of the endogenous SBF DNA-binding domain (DBD) with that from more distantly related fungi leads to a contraction of the SBF-regulated G1/S network in budding yeast, which also correlates with increased defects in cell growth, cell size, and proliferation.

The Role of Hsp70 in the Regulation of Autophagy in Gametogenesis, Pregnancy, and Parturition.

Induction of the 70kDa heat shock protein (hsp70) and autophagy are two major mechanisms that promote cell homeostasis during the rapid cell growth and differentiation characteristic of reproduction. Hsp70 insures proper assembly, conformation, and intracellular transport of nascent proteins. Autophagy removes from the cytoplasm proteins, other macromolecules, and organelles that are no longer functional or needed and recycles their components for synthesis of new products under nutritionally limiting conditions. Hsp70 inhibits autophagy and so a proper balance between these two processes is essential for optimal germ cell production and survival and pregnancy progression. A marked inhibition in autophagy and a concomitant increase in hsp70 at term is a trigger for parturition. Excessive external or endogenous stress that induces a high level of hsp70 production can lead to a non-physiological inhibition of autophagy, resulting in altered spermatogenesis, premature ovarian failure, and complications of pregnancy including preeclampsia, intrauterine growth restriction, and preterm birth.

Remodeling Ca2+ Flux By ORP4L Is Essential for Leukemia Stem Cells (LSCs) Survival

Acute myelogenous leukemia (AML) is one of the deadliest hematological malignancies and there is at present no efficient strategy to defeat it. New detailed insight into AML leukemia stem cells (LSCs) survival will facilitate the identification of targets for the development of new therapeutic approaches. Recent work has provided evidence that LSCs are defective in their ability to employ glycolysis, but are highly reliant on oxidative phosphorylation, and the maintenance of mitochondrial function is essential for LSCs survival. It is increasingly clear that Ca2+ released constitutively from endoplasmic reticulum (ER) is taken up by mitochondria to sustain optimal bioenergetics and cell survival. Here we report three striking findings: 1) oxysterol-binding protein (OSBP)-related protein 4 (ORP4L) is expressed in LSCs but not in normal hematopoietic stem cells (HSCs). 2) ORP4L is essential for LSC bioenergetics; It forms a complex with PLCβ3 and IP3 receptor 1 (ITPR1) to control Ca2+ release from the ER and subsequent cytosolic and mitochondrial parallel Ca2+ spike oscillations that sustain pyruvate dehydrogenase (PDH) activation and oxidative phosphorylation. 3) ORP4L inhibition eradicates LSCs in vitro and in vivo through impairment of Ca2+-dependent bioenergetics. These results suggest a novel role of ORP4L in governing Ca2+ release to sustain mitochondrial function and survival of LSCs and identify ORP4L as a putative new oncoprotein and therapeutic target for LSCs elimination. DisclosuresNo relevant conflicts of interest to declare.

What Kind of Signaling Maintains Pluripotency and Viability in Human-Induced Pluripotent Stem Cells Cultured on Laminin-511 with Serum-Free Medium?

Xeno-free medium contains no animal-derived components, but is composed of minimal growth factors and is serum free; the medium may be supplemented with insulin, transferrin, and selenium (ITS medium). Serum-free and xeno-free culture of human-induced pluripotent stem cells (hiPSCs) uses a variety of components based on ITS medium and Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (DMEM/F12) that contain high levels of iron salt and glucose. Culture of hiPSCs also requires scaffolding materials, such as extracellular matrix, collagen, fibronectin, laminin, proteoglycan, and vitronectin. The scaffolding component laminin-511, which is composed of α5, β1, and γ1 chains, binds to α3β1, α6β1, and α6β4 integrins on the cell membrane to induce activation of the PI3K/AKT- and Ras/MAPK-dependent signaling pathways. In hiPSCs, the interaction of laminin-511/α6β1 integrin with the cell–cell adhesion molecule E-cadherin confers protection against apoptosis through the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathways for cell death) and the proto-oncogene tyrosine-protein kinase Fyn (Fyn)-RhoA-ROCK signaling pathway. The expression levels of α6β1 integrin and E-cadherin on cell membranes are controlled through the activation of insulin receptor/insulin, FGF receptor/FGF2, or activin-like kinase 5 (ALK5)-dependent TGF-β signaling. A combination of growth factors, medium constituents, cell membrane-located E-cadherin, and α6β1 integrin-induced signaling is required for pluripotent cell proliferation and for optimal cell survival on a laminin-511 scaffold. In this review, we discuss and explore the influence of growth factors on the cadherin and integrin signaling pathways in serum-free and xeno-free cultures of hiPSCs during the preparation of products for regenerative medicinal therapies. In addition, we suggest the optimum serum-free medium components for use with laminin-511, a new scaffold for hiPSC culture.

The Unfolded Protein Response Triggers Site-Specific Regulatory Ubiquitylation of 40S Ribosomal Proteins

Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as an important facet of eukaryotic translational control.

Survival of skin cancer stem cells requires the Ezh2 polycomb group protein.

Polycomb group proteins, including Ezh2, are important candidate stem cell maintenance proteins in epidermal squamous cell carcinoma. We previously showed that epidermal cancer stem cells (ECS cells) represent a minority of cells in tumors, are highly enriched in Ezh2 and drive aggressive tumor formation. We now show that Ezh2 is required for ECS cell survival, migration, invasion and tumor formation and that this is associated with increased histone H3 trimethylation on lysine 27, a mark of Ezh2 action. We also show that Ezh2 knockdown or treatment with Ezh2 inhibitors, GSK126 or EPZ-6438, reduces Ezh2 level and activity, leading to reduced ECS cell spheroid formation, migration, invasion and tumor growth. These studies indicate that epidermal squamous cell carcinoma cells contain a subpopulation of cancer stem (tumor-initiating) cells that are enriched in Ezh2, that Ezh2 is required for optimal ECS cell survival and tumor formation and that treatment with Ezh2 inhibitors may be a strategy for reducing ECS cell survival and suppressing tumor formation.

The combined loss of Gads and CD127 reveals a novel function of Gads prior to TCRβ expression.

The Gads adaptor protein is an essential component of the T cell signaling complex critical for T cell receptor-mediated calcium mobilization. After expression of TCRβ in T cell precursors, Gads is required for optimal Bcl-2 expression and cell survival. Similarly, the IL-7 receptor chain CD127 is also necessary for optimal Bcl-2 expression and cell survival in TCRβ-expressing thymocytes. Based on these observations, we tested whether Gads and CD127 might regulate convergent or linear signaling pathways by crossing Gads(-/-) mice with CD127(-/-) mice. Thymi from Gads(-/-)CD127(-/-) mice were barely detectable and many of the thymocytes were within the DN1 population. By contrast, B cell development in the Gads(-/-)CD127(-/-) mice was comparable to that of CD127(-/-) mice, indicating that the combined loss of Gads and CD127 did not lead to a global deficit in hematopoiesis. Analysis of Lin(-)Sca-1(+)c-kit(+) bone marrow cells and bone marrow chimera experiments indicated that Gads(-/-)CD127(-/-) T cell precursors either failed to migrate into the thymus or survive in the thymus. These data demonstrate that Gads functions at a stage of T cell development that had not been previously described.

RGD‐peptide modified alginate by a chemoenzymatic strategy for tissue engineering applications

One of the main challenges in tissue engineering and regenerative medicine is the ability to maintain optimal cell function and survival post-transplantation. Biomaterials such as alginates are commonly used for immunoisolation, while they may also provide structural support to the cell transplants by mimicking the extracellular matrix. In this study, arginine-glycine-aspartate (RGD)-peptide-coupled alginates of tailored composition were produced by adopting a unique chemoenzymatic strategy for substituting the nongelling mannuronic acid on the alginate. Alginates with and without RGD were produced with high and low content of G. Using carbodiimide chemistry 0.1-0.2% of the sugar units were substituted by peptide. Furthermore, the characterization by (1)H-nuclear magnetic resonance (NMR) revealed by-products from the coupling reaction that partly could be removed by coal filtration. Olfactory ensheathing cells (OECs) and myoblasts were grown in two-dimensional (2D) and 3D cultures of RGD-peptide modified or unmodified alginates obtained by the chemoenzymatically strategy and compared to native alginate. Both OECs and myoblasts adhered to the RGD-peptide modified alginates in 2D cultures, forming bipolar protrusions. OEC encapsulation resulted in cell survival for up to 9 days, thus demonstrating the potential for short-term 3D culture. Myoblasts showed long-term survival in 3D cultures, that is, up to 41 days post encapsulation. The RGD modifications did not result in marked changes in cell viability in 3D cultures. We demonstrate herein a unique technique for tailoring peptide substituted alginates with a precise and flexible composition, conserving the gel forming properties relevant for the use of alginate in tissue engineering.

Improving survival and efficacy of pluripotent stem cell–derived cardiac grafts

Human embryonic stem cells (hESCs) can be differentiated into structurally and electrically functional myocardial tissue and have the potential to regenerate large regions of infarcted myocardium. One of the key challenges that needs to be addressed towards full-scale clinical application of hESCs is enhancing survival of the transplanted cells within ischaemic or scarred, avascular host tissue. Shortly after transplantation, most hESCs are lost as a result of multiple mechanical, cellular and host factors, and a large proportion of the remaining cells undergo apoptosis or necrosis shortly thereafter, as a result of loss of adhesion-related signals, ischaemia, inflammation or immunological rejection. Blocking the apoptotic signalling pathways of the cells, using pro-survival cocktails, conditioning hESCs prior to transplant, promoting angiogenesis, immunosuppressing the host and using of bioengineered matrices are among the emerging techniques that have been shown to optimize cell survival. This review presents an overview of the current strategies for optimizing cell and host tissue to improve the survival and efficacy of cardiac cells derived from pluripotent stem cells.