Cadherin-11 Influences Differentiation in Human Mesenchymal Stem Cells by Regulating the Extracellular Matrix Via the TGFß1 Pathway.

For regenerative medicine, directing stem cell fate is one of the key aims. Human mesenchymal stem cells (hMSCs) are versatile adult stem cells that have been proposed for several clinical applications, making directing their fate of utmost importance. For most clinical applications, their differentiation toward the adipogenic lineage is an undesired outcome. Understanding the mechanisms that regulate hMSC commitment toward the adipogenic lineage might help open up new avenues for fine-tuning implanted hMSCs for regenerative medicine applications. We know that cadherin-11 is required for hMSC commitment to the adipogenic lineage; therefore, we sought to investigate the mechanisms through which cadherin-11 regulates adipogenic differentiation. We observed that hMSCs lacking cadherin-11 had decreased expression of type VI collagen and increased expression of fibronectin. We provide evidence of increased transforming growth factor beta 1 and the subsequent translocation of phosphorylated SMAD2/3 into the nucleus by cells that lack cadherin-11, which could be attributed to the changes in extracellular matrix composition. Taken together, our study implicates cadherin-11 in regulating extracellular matrix production and thereby helping improve cell- and material-based regenerative medicine approaches.

Vascular bioengineering of scaffolds derived from human discarded transplant kidneys using human pluripotent stem cell-derived endothelium.

The bioengineering of a replacement kidney has been proposed as an approach to address the growing shortage of donor kidneys for the treatment of chronic kidney disease. One approach being investigated is the recellularization of kidney scaffolds. In this study, we present several key advances toward successful re-endothelialization of whole kidney matrix scaffolds from both rodents and humans. Based on the presence of preserved glycosoaminoglycans within the decelullarized kidney scaffold, we show improved localization of delivered endothelial cells after preloading of the vascular matrix with vascular endothelial growth factor and angiopoietin 1. Using a novel simultaneous arteriovenous delivery system, we report the complete re-endothelialization of the kidney vasculature, including the glomerular and peritubular capillaries, using human inducible pluripotent stem cell -derived endothelial cells. Using this source of endothelial cells, it was possible to generate sufficient endothelial cells to recellularize an entire human kidney scaffold, achieving efficient cell delivery, adherence, and endothelial cell proliferation and survival. Moreover, human re-endothelialized scaffold could, in contrast to the non-re-endothelialized human scaffold, be fully perfused with whole blood. These major advances move the field closer to a human bioengineered kidney.

Novel insights in the disease biology of mutant small heat shock proteins in neuromuscular diseases.

Small heat shock proteins are molecular chaperones that exert diverse cellular functions. To date, mutations in the coding regions of HSPB1 (Hsp27) and HSPB8 (Hsp22) were reported to cause distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. Recently, the clinical spectrum of HSPB1 and HSPB8 mutations was expanded to also include myopathies. Here we provide an update on the molecular genetics and biology of small heat shock protein mutations in neuromuscular diseases.

Axonal Neuropathies due to Mutations in Small Heat Shock Proteins: Clinical, Genetic, and Functional Insights into Novel Mutations.

In this study, we describe the phenotypic spectrum of distal hereditary motor neuropathy caused by mutations in the small heat shock proteins HSPB1 and HSPB8 and investigate the functional consequences of newly discovered variants. Among 510 unrelated patients with distal motor neuropathy, we identified mutations in HSPB1 (28 index patients/510; 5.5%) and HSPB8 (four index patients/510; 0.8%) genes. Patients have slowly progressive distal (100%) and proximal (13%) weakness in lower limbs (100%), mild lower limbs sensory involvement (31%), foot deformities (73%), progressive distal upper limb weakness (29%), mildly raised serum creatine kinase levels (100%), and central nervous system involvement (9%). We identified 12 HSPB1 and four HSPB8 mutations, including five and three not previously reported. Transmission was either dominant (78%), recessive (3%), or de novo (19%). Three missense mutations in HSPB1 (Pro7Ser, Gly53Asp, and Gln128Arg) cause hyperphosphorylation of neurofilaments, whereas the C-terminal mutant Ser187Leu triggers protein aggregation. Two frameshift mutations (Leu58fs and Ala61fs) create a premature stop codon leading to proteasomal degradation. Two mutations in HSPB8 (Lys141Met/Asn) exhibited increased binding to Bag3. We demonstrate that HSPB1 and HSPB8 mutations are a major cause of inherited motor axonal neuropathy. Mutations lead to diverse functional outcomes further demonstrating the pleotropic character of small heat shock proteins.

Molecular Chaperones in the Pathogenesis of Amyotrophic Lateral Sclerosis: The Role of HSPB1.

Genetic discoveries in amyotrophic lateral sclerosis (ALS) have a significant impact on deciphering molecular mechanisms of motor neuron degeneration but, despite recent advances, the etiology of most sporadic cases remains elusive. Several cellular mechanisms contribute to the motor neuron degeneration in ALS, including RNA metabolism, cellular interactions between neurons and nonneuronal cells, and seeding of misfolded protein with prion-like propagation. In this scenario, the importance of protein turnover and degradation in motor neuron homeostasis gained increased recognition. In this study, we evaluated the role of the candidate gene HSPB1, a molecular chaperone involved in several proteome-maintenance functions. In a cohort of 247 unrelated Italian ALS patients, we identified two variants (c.570G>C, p.Gln190His and c.610dupG, p.Ala204Glyfs* 6). Functional characterization of the p.Ala204Glyfs* 6 demonstrated that the mutant protein alters HSPB1 dynamic equilibrium, sequestering the wild-type protein in a stable dimer and resulting in a loss of chaperone-like activity. Our results underline the relevance of identifying rare but pathogenic variations in sporadic neurodegenerative diseases, suggesting a possible correlation between specific pathomechanisms linked to HSPB1 mutations and the associated neurological phenotype. Our study provides additional lines of evidence to support the involvement of HSPB1 in the pathogenesis of sporadic ALS.

The hnRNP family: insights into their role in health and disease.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins (RBPs) that contribute to multiple aspects of nucleic acid metabolism including alternative splicing, mRNA stabilization, and transcriptional and translational regulation. Many hnRNPs share general features, but differ in domain composition and functional properties. This review will discuss the current knowledge about the different hnRNP family members, focusing on their structural and functional divergence. Additionally, we will highlight their involvement in neurodegenerative diseases and cancer, and the potential to develop RNA-based therapies.

Molecular defects in the motor adaptor BICD2 cause proximal spinal muscular atrophy with autosomal-dominant inheritance.

The most common form of spinal muscular atrophy (SMA) is a recessive disorder caused by deleterious SMN1 mutations in 5q13, whereas the genetic etiologies of non-5q SMA are very heterogeneous and largely remain to be elucidated. In a Bulgarian family affected by autosomal-dominant proximal SMA, we performed genome-wide linkage analysis and whole-exome sequencing and found a heterozygous de novo c.320C>T (p.Ser107Leu) mutation in bicaudal D homolog 2 (Drosophila) (BICD2). Further analysis of BICD2 in a cohort of 119 individuals with non-5q SMA identified a second de novo BICD2 mutation, c.2321A>G (p.Glu774Gly), in a simplex case. Detailed clinical and electrophysiological investigations revealed that both families are affected by a very similar disease course, characterized by early childhood onset, predominant involvement of lower extremities, and very slow disease progression. The amino acid substitutions are located in two interaction domains of BICD2, an adaptor protein linking the dynein molecular motor with its cargo. Our immunoprecipitation and localization experiments in HeLa and SH-SY5Y cells and affected individuals' lymphoblasts demonstrated that p.Ser107Leu causes increased dynein binding and thus leads to accumulation of BICD2 at the microtubule-organizing complex and Golgi fragmentation. In addition, the altered protein had a reduced colocalization with RAB6A, a regulator of vesicle trafficking between the Golgi and the endoplasmic reticulum. The interaction between p.Glu744Gly altered BICD2 and RAB6A was impaired, which also led to their reduced colocalization. Our study identifies BICD2 mutations as a cause of non-5q linked SMA and highlights the importance of dynein-mediated motility in motor neuron function in humans.

Recessive mutations in SLC13A5 result in a loss of citrate transport and cause neonatal epilepsy, developmental delay and teeth hypoplasia.

The epileptic encephalopathies are a clinically and aetiologically heterogeneous subgroup of epilepsy syndromes. Most epileptic encephalopathies have a genetic cause and patients are often found to carry a heterozygous de novo mutation in one of the genes associated with the disease entity. Occasionally recessive mutations are identified: a recent publication described a distinct neonatal epileptic encephalopathy (MIM 615905) caused by autosomal recessive mutations in the SLC13A5 gene. Here, we report eight additional patients belonging to four different families with autosomal recessive mutations in SLC13A5. SLC13A5 encodes a high affinity sodium-dependent citrate transporter, which is expressed in the brain. Neurons are considered incapable of de novo synthesis of tricarboxylic acid cycle intermediates; therefore they rely on the uptake of intermediates, such as citrate, to maintain their energy status and neurotransmitter production. The effect of all seven identified mutations (two premature stops and five amino acid substitutions) was studied in vitro, using immunocytochemistry, selective western blot and mass spectrometry. We hereby demonstrate that cells expressing mutant sodium-dependent citrate transporter have a complete loss of citrate uptake due to various cellular loss-of-function mechanisms. In addition, we provide independent proof of the involvement of autosomal recessive SLC13A5 mutations in the development of neonatal epileptic encephalopathies, and highlight teeth hypoplasia as a possible indicator for SLC13A5 screening. All three patients who tried the ketogenic diet responded well to this treatment, and future studies will allow us to ascertain whether this is a recurrent feature in this severe disorder.

The response of three-dimensional pancreatic alpha and beta cell co-cultures to oxidative stress.

The pancreatic islets of Langerhans have low endogenous antioxidant levels and are thus especially sensitive to oxidative stress, which is known to influence cell survival and behaviour. As bioengineered islets are gaining interest for therapeutic purposes, it is important to understand how their composition can be optimized to diminish oxidative stress. We investigated how the ratio of the two main islet cell types (alpha and beta cells) and their culture in three-dimensional aggregates could protect against oxidative stress. Monolayer and aggregate cultures were established by seeding the alphaTC1 (alpha) and INS1E (beta) cell lines in varying ratios, and hydrogen peroxide was applied to induce oxidative stress. Viability, oxidative stress, and the level of the antioxidant glutathione were measured. Both aggregation and an increasing prevalence of INS1E cells in the co-cultures conferred greater resistance to cell death induced by oxidative stress. Increasing the prevalence of INS1E cells also decreased the number of alphaTC1 cells experiencing oxidative stress in the monolayer culture. In 3D aggregates, culturing the alphaTC1 and INS1E cells in a ratio of 50:50 prevented oxidative stress in both cell types. Together, the results of this study lead to new insight into how modulating the composition and dimensionality of a co-culture can influence the oxidative stress levels experienced by the cells.

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells.

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10-R98S). Our data support that RPL10-R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock-in of RPL10-R98S was associated with a 1.7- to 2.5-fold increased production of four transiently expressed recombinant proteins and 1.7-fold for one out of two stably expressed proteins. In CHO-S cells, eGFP reached a 2-fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10-R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10-R98S phenotypes can maximize its exploitability for the mammalian protein production industry.

Sticking together: Harnessing cadherin biology for tissue engineering.

Directing cell behavior and building a tissue for therapeutic impact is the main goal of regenerative medicine, for which scientists need to modulate the interaction of cells with biomaterials. The focus of the field thus far has been on the incorporation of cues from the extracellular matrix but we propose that scientists take lessons from cell-cell adhesion proteins, more specifically cadherin biology, as these proteins make multicellularity possible. In this perspective, we re-examine cadherins through the lens of a tissue engineer for the purpose of advancing regenerative medicine. Furthermore, we summarize exciting developments in biomaterials inspired by cadherins and discuss some challenges and opportunities for the future. STATEMENT OF SIGNIFICANCE: Tissue engineers need tools to direct cell behavior. To date, tissue engineers have designed many sophisticated materials to positively influence cell behavior but are faced with the challenge where these materials sometimes work and sometimes fail. This uncertainty is a big unanswered question that challenges the community. We propose that tissue engineering could be more successful if they would take lessons from cell-cell adhesion proteins, more specifically cadherin biology. In the article, we discuss key structural and functional characteristics that make cadherins ideal for tissue engineering approaches. Furthermore, by providing a state-of-the-art overview of exemplary studies that have used cadherins to influence cell behavior, we show tissue engineers that they already have the tools necessary to incorporate this knowledge.

Win, Lose, or Tie: Mathematical Modeling of Ligand Competition at the Cell-Extracellular Matrix Interface.

Integrin transmembrane proteins conduct mechanotransduction at the cell-extracellular matrix (ECM) interface. This process is central to cellular homeostasis and therefore is particularly important when designing instructive biomaterials and organoid culture systems. Previous studies suggest that fine-tuning the ECM composition and mechanical properties can improve organoid development. Toward the bigger goal of fully functional organoid development, we hypothesize that resolving the dynamics of ECM-integrin interactions will be highly instructive. To this end, we developed a mathematical model that enabled us to simulate three main interactions, namely integrin activation, ligand binding, and integrin clustering. Different from previously published computational models, we account for the binding of more than one type of ligand to the integrin. This competition between ligands defines the fate of the system. We have demonstrated that an increase in the initial concentration of ligands does not ensure an increase in the steady state concentration of ligand-bound integrins. The ligand with higher binding rate occupies more integrins at the steady state than does the competing ligand. With cell type specific, quantitative input on integrin-ligand binding rates, this model can be used to develop instructive cell culture systems.

Corrigendum: Win, Lose or Tie: Mathematical Modeling of Ligand Competition at the Cell-Extracellular Matrix Interface.

[This corrects the article DOI: 10.3389/fbioe.2021.657244.].

The Role of Pancreatic Alpha Cells and Endothelial Cells in the Reduction of Oxidative Stress in Pseudoislets.

Pancreatic beta cells have inadequate levels of antioxidant enzymes, and the damage induced by oxidative stress poses a challenge for their use in a therapy for patients with type 1 diabetes. It is known that the interaction of the pancreatic endocrine cells with support cells can improve their survival and lead to less vulnerability to oxidative stress. Here we investigated alpha (alpha TC-1), beta (INS1E) and endothelial (HUVEC) cells assembled into aggregates known as pseudoislets as a model of the pancreatic islets of Langerhans. We hypothesised that the coculture of alpha, beta and endothelial cells would be protective against oxidative stress. First, we showed that adding endothelial cells decreased the percentage of oxidative stress-positive cells. We then asked if the number of endothelial cells or the size (number of cells) of the pseudoislet could increase the protection against oxidative stress. However, no additional benefit was observed by those changes. On the other hand, we identified a potential supportive effect of the alpha cells in reducing oxidative stress in beta and endothelial cells. We were able to link this to the incretin glucagon-like peptide-1 (GLP-1) by showing that the absence of alpha cells in the pseudoislet caused increased oxidative stress, but the addition of GLP-1 could restore this. Together, these results provide important insights into the roles of alpha and endothelial cells in protecting against oxidative stress.

The Role of Alpha Cells in the Self-Assembly of Bioengineered Islets.

Vascularization is undoubtedly one of the greatest challenges in tissue engineering. Its importance is particularly evident when considering the transplantation of (bioengineered) pancreatic islets of Langerhans, which are highly sensitive to the delivery of oxygen and nutrients for their survival and function. Here we studied pseudoislets of Langerhans, which are three-dimensional spheroids composed of ß (INS1E), α (alpha TC-1), and endothelial (HUVEC) cells, and were interested in how the location and prevalence of the different cell types affected the presence of endothelial cells in the pseudoislet. We hypothesized that alpha (α) cells play an essential role in islet self-assembly and the incorporation of endothelial cells into the pseudoislet, and are thus important to consider in tissue engineering or regenerative medicine strategies, which typically focuses on the insulin-producing beta (ß) cells alone. We first determined the effect of changing the relative ratios of the cells and found the cell distribution converged on a steady state of ∼21% α cells, 74% ß cells, and 5% endothelial cells after 10 days of culture regardless of their respective ratios at seeding. We also found that the incorporation of endothelial cells was related to the pseudoislet size, with more endothelial cells found in the core of larger pseudoislets following a concomitant increase of α cells and a decrease in ß cells. Finally, we observed that both endothelial and ß cells were found adjacent to α cells significantly more frequently than to each other. In conclusion, this study demonstrates that the self-assembly of a pseudoislet is an intrinsically cell-regulated process. The endothelial cells had preferential proximity to the α cells, and this persisted even when challenged with changing the cell ratios and numbers. This study gives insight into the rules governing the self-organization of pseudoislets and suggests an important role for α cells to promote the incorporation of endothelial cells.

Thiol-ene cross-linked alginate hydrogel encapsulation modulates the extracellular matrix of kidney organoids by reducing abnormal type 1a1 collagen deposition.

Differentiated kidney organoids from induced pluripotent stem cells hold promise as a treatment for patients with kidney diseases. Before these organoids can be translated to the clinic, shortcomings regarding their cellular and extracellular compositions, and their developmental plateau need to be overcome. We performed a proteomic analysis on kidney organoids cultured for a prolonged culture time and we found a specific change in the extracellular matrix composition with increased expression of types 1a1, 2 and 6a1 collagen. Such an excessive accumulation of specific collagen types is a hallmark of renal fibrosis that causes a life-threatening pathological condition by compromising key functions of the human kidney. Here we hypothesized the need for a three-dimensional environment to grow the kidney organoids, which could better mimic the in vivo surroundings of the developing kidney than standard culture on an air-liquid interface. Encapsulating organoids for four days in a soft, thiol-ene cross-linked alginate hydrogel resulted in decreased type 1a1 collagen expression. Furthermore, the encapsulation did not result in any changes of organoid structural morphology. Using a biomaterial to modulate collagen expression allows for a prolonged kidney organoid culture in vitro and a reduction of abnormal type 1a1 collagen expression bringing kidney organoids closer to clinical application.

Overcoming kidney organoid challenges for regenerative medicine.

Kidney organoids derived from human induced pluripotent stem cells bear the potential to be used as a regenerative medicine renal replacement therapy. Advances in developmental biology shed light on the complex cellular regulation during kidney morphogenesis in animal models resulting in insights that were incorporated in the development of groundbreaking protocols for the directed differentiation of human pluripotent stem cells to kidney endpoints. Moreover, further optimization efforts to improve three-dimensional culture techniques resulted in the creation of kidney organoids. Before they can find their way to the clinic, there are critical challenges to overcome. Here, we will discuss recent advances and remaining challenges for kidney organoids to become successful in regenerative medicine. An innovative combination of tissue engineering techniques with more refined insights in the developing human kidney will ultimately lead to more mature and functional kidney organoids suitable as renal replacement therapy for patients with chronic kidney disease.

Cell culture dimensionality influences mesenchymal stem cell fate through cadherin-2 and cadherin-11.

The acquisition of a specific cell fate is one of the core aims of tissue engineering and regenerative medicine. Significant evidence shows that aggregate cultures have a positive influence on cell fate decisions, presumably through cell-cell interactions, but little is known about the specific mechanisms. To investigate the difference between cells cultured as a monolayer and as aggregates, we started by looking at cadherin expression, an important protein involved in cell adhesion, during the differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in aggregate and monolayer cultures. We observed that proliferating hMSCs in monolayer culture expressed lower levels of cadherin-2 and increased cadherin-11 expression at cell-cell contact sites over time, which was not evident in the aggregate cultures. By knocking down cadherin-2 and cadherin-11, we found that both cadherins were required for adipogenic differentiation in a monolayer as well as aggregate culture. However, during osteogenic differentiation, low levels of cadherin-2 were found to be favorable for cells cultured as a monolayer and as aggregates, whereas cadherin- 11 was dispensable for cells cultured as aggregates. Together, these results provide compelling evidence for the important role that cadherins play in regulating the differentiation of hMSCs and how this is affected by the dimensionality of cell culture.

Mutant HSPB1 causes loss of translational repression by binding to PCBP1, an RNA binding protein with a possible role in neurodegenerative disease.

The small heat shock protein HSPB1 (Hsp27) is an ubiquitously expressed molecular chaperone able to regulate various cellular functions like actin dynamics, oxidative stress regulation and anti-apoptosis. So far disease causing mutations in HSPB1 have been associated with neurodegenerative diseases such as distal hereditary motor neuropathy, Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis. Most mutations in HSPB1 target its highly conserved α-crystallin domain, while other mutations affect the C- or N-terminal regions or its promotor. Mutations inside the α-crystallin domain have been shown to enhance the chaperone activity of HSPB1 and increase the binding to client proteins. However, the HSPB1-P182L mutation, located outside and downstream of the α-crystallin domain, behaves differently. This specific HSPB1 mutation results in a severe neuropathy phenotype affecting exclusively the motor neurons of the peripheral nervous system. We identified that the HSPB1-P182L mutant protein has a specifically increased interaction with the RNA binding protein poly(C)binding protein 1 (PCBP1) and results in a reduction of its translational repressive activity. RNA immunoprecipitation followed by RNA sequencing on mouse brain lead to the identification of PCBP1 mRNA targets. These targets contain larger 3'- and 5'-UTRs than average and are enriched in an RNA motif consisting of the CTCCTCCTCCTCC consensus sequence. Interestingly, next to the clear presence of neuronal transcripts among the identified PCBP1 targets we identified known genes associated with hereditary peripheral neuropathies and hereditary spastic paraplegias. We therefore conclude that HSPB1 can mediate translational repression through interaction with an RNA binding protein further supporting its role in neurodegenerative disease.

Characterization of New Transgenic Mouse Models for Two Charcot-Marie-Tooth-Causing HspB1 Mutations using the Rosa26 Locus.

BACKGROUND: Charcot-Marie-Tooth (CMT) and associated neuropathies, the most common inherited diseases of the peripheral nervous system, remain so far incurable. Three existing murine models of Charcot-Marie-Tooth type 2F (CMT2F) and/or distal hereditary motor neuropathy type IIb (dHMNIIb), caused by mutations in the small heat shock protein B1 gene (HSPB1/HSP27), partially recapitulate the hallmarks of peripheral neuropathy. Because these models overexpress the HSPB1 mutant proteins they differ from the patients' situation. OBJECTIVE: To overcome the possible bias induced by overexpression, we generated and characterized a transgenic model in which the wild type or mutant HSPB1 protein was expressed at a moderate, more physiologically relevant level. METHODS: We generated a new transgenic mouse model in which a human wild type (hHSPB1WT) or mutant (hHSPB1R127W; hHSPB1P182L) HSPB1 transgene was integrated in the mouse ROSA26 locus. The motor and sensory functions of the mice was assessed at 3, 6, 9, 12 and 18 month. RESULTS: However, the mice expressing the mutant hHSPB1 do not develop motor or sensory deficits and do not show any sign of axonal degeneration, even at late age. Quantitative PCR analyses reveal contrasting tissue-specific expression pattern for the endogenous mouse and exogenous human HSPB1 and show that the ratio of human HSPB1 to the endogenous mouse HspB1 is lower in the sciatic nerve and spinal cord compared to the brain. CONCLUSION: These results suggest that expressing the transgene at a physiological level using the ROSA26 locus may not be sufficient to model inherited peripheral neuropathies caused by mutation in HSPB1.