Monochorionic Twinning in Bioengineered Human Embryo Models.

Monochorionic twinning of human embryos increases the risk of complications during pregnancy. The rarity of such twinning events, combined with ethical constraints in human embryo research, makes investigating the mechanisms behind twinning practically infeasible. As a result, there is a significant knowledge gap regarding the origins and early phenotypic presentation of monochorionic twin embryos. In this study, a microthermoformed-based microwell screening platform is used to identify conditions that efficiently induce monochorionic twins in human stem cell-based blastocyst models, termed "twin blastoids". These twin blastoids contain a cystic GATA3+ trophectoderm-like epithelium encasing two distinct inner cell masses (ICMs). Morphological and morphokinetic analyses reveal that twinning occurs during the cavitation phase via splitting of the OCT4+ pluripotent core. Notably, each ICM in twin blastoids contains its own NR2F2+ polar trophectoderm-like region, ready for implantation. This is functionally tested in a microfluidic chip-based implantation assay with epithelial endometrium cells. Under defined flow regimes, twin blastoids show increased adhesion capacity compared to singleton blastoids, suggestive of increased implantation potential. In conclusion, the development of technology enabling large-scale formation of twin blastoids, coupled with high-sensitivity readout capabilities, presents an unprecedented opportunity for systematically exploring monochorionic twin formation and its impact on embryonic development.

Multipotent adult progenitor cells prevent functional impairment and improve development in inflammation driven detriment of preterm ovine lungs.

Background: Perinatal inflammation increases the risk for bronchopulmonary dysplasia in preterm neonates, but the underlying pathophysiological mechanisms remain largely unknown. Given their anti-inflammatory and regenerative capacity, multipotent adult progenitor cells (MAPC) are a promising cell-based therapy to prevent and/or treat the negative pulmonary consequences of perinatal inflammation in the preterm neonate. Therefore, the pathophysiology underlying adverse preterm lung outcomes following perinatal inflammation and pulmonary benefits of MAPC treatment at the interface of prenatal inflammatory and postnatal ventilation exposures were elucidated. Methods: Instrumented ovine fetuses were exposed to intra-amniotic lipopolysaccharide (LPS 5 mg) at 125 days gestation to induce adverse systemic and peripheral organ outcomes. MAPC (10 × 106 cells) or saline were administered intravenously two days post LPS exposure. Fetuses were delivered preterm five days post MAPC treatment and either killed humanely immediately or mechanically ventilated for 72 h. Results: Antenatal LPS exposure resulted in inflammation and decreased alveolar maturation in the preterm lung. Additionally, LPS-exposed ventilated lambs showed continued pulmonary inflammation and cell junction loss accompanied by pulmonary edema, ultimately resulting in higher oxygen demand. MAPC therapy modulated lung inflammation, prevented loss of epithelial and endothelial barriers and improved lung maturation in utero. These MAPC-driven improvements remained evident postnatally, and prevented concomitant pulmonary edema and functional loss. Conclusion: In conclusion, prenatal inflammation sensitizes the underdeveloped preterm lung to subsequent postnatal inflammation, resulting in injury, disturbed development and functional impairment. MAPC therapy partially prevents these changes and is therefore a promising approach for preterm infants to prevent adverse pulmonary outcomes.

A pendulum of induction between the epiblast and extra-embryonic endoderm supports post-implantation progression.

Embryogenesis is supported by dynamic loops of cellular interactions. Here, we create a partial mouse embryo model to elucidate the principles of epiblast (Epi) and extra-embryonic endoderm co-development (XEn). We trigger naive mouse embryonic stem cells to form a blastocyst-stage niche of Epi-like cells and XEn-like cells (3D, hydrogel free and serum free). Once established, these two lineages autonomously progress in minimal medium to form an inner pro-amniotic-like cavity surrounded by polarized Epi-like cells covered with visceral endoderm (VE)-like cells. The progression occurs through reciprocal inductions by which the Epi supports the primitive endoderm (PrE) to produce a basal lamina that subsequently regulates Epi polarization and/or cavitation, which, in return, channels the transcriptomic progression to VE. This VE then contributes to Epi bifurcation into anterior- and posterior-like states. Similarly, boosting the formation of PrE-like cells within blastoids supports developmental progression. We argue that self-organization can arise from lineage bifurcation followed by a pendulum of induction that propagates over time.

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.

Synthetic Materials that Affect the Extracellular Matrix via Cellular Metabolism and Responses to a Metabolic State.

In regenerative medicine and tissue engineering, many materials are developed to mimic the extracellular matrix (ECM). However, these ECM-mimicking materials do not yet completely recapitulate the diversity and complexity of biological tissue-specific ECM. In this review, an alternative strategy is proposed to generate ECM, namely synthesizing a material that functions as a drug delivery system, releasing molecules that target cellular metabolic pathways and thereby stimulate the local cells to create their own ECM. This is based on the fact that ECM synthesis, modification, composition, signaling, stiffness, and degradation are modulated by cellular metabolism. Metabolism can be targeted at different levels, ranging from modulating the availability of substrates or co-factors to regulating the activity of essential transcription factors. Depending on the drug of interest, its characteristics, mechanism of action, cellular target, and application, a different drug delivery system should be designed. Metabolic drugs modulating the ECM require cellular uptake for their function, therefore reversible linkers are recommended. Preferably the metabolic modulators are only released when needed, which will be upon a specific metabolic state, a change in ECM stiffness, or ECM remodeling. Therefore, reversible linkers that respond to an environmental stimulus could be incorporated. All in all, a novel strategy is suggested to develop a tissue-specific ECM by generating a synthetic material that releases metabolic molecules modulating the ECM. Various ways to modulate the ECM properties via the metabolism are reviewed and guidelines for the development of these materials are provided.

PEOT/PBT Polymeric Pastes to Fabricate Additive Manufactured Scaffolds for Tissue Engineering.

The advantages of additive manufactured scaffolds, as custom-shaped structures with a completely interconnected and accessible pore network from the micro- to the macroscale, are nowadays well established in tissue engineering. Pore volume and architecture can be designed in a controlled fashion, resulting in a modulation of scaffold's mechanical properties and in an optimal nutrient perfusion determinant for cell survival. However, the success of an engineered tissue architecture is often linked to its surface properties as well. The aim of this study was to create a family of polymeric pastes comprised of poly(ethylene oxide therephthalate)/poly(butylene terephthalate) (PEOT/PBT) microspheres and of a second biocompatible polymeric phase acting as a binder. By combining microspheres with additive manufacturing technologies, we produced 3D scaffolds possessing a tailorable surface roughness, which resulted in improved cell adhesion and increased metabolic activity. Furthermore, these scaffolds may offer the potential to act as drug delivery systems to steer tissue regeneration.

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.

Oxidative stress in pancreatic alpha and beta cells as a selection criterion for biocompatible biomaterials.

The clinical success rate of islet transplantation, namely independence from insulin injections, is limited by factors that lead to graft failure, including inflammation, acute ischemia, acute phase response, and insufficient vascularization. The ischemia and insufficient vascularization both lead to high levels of oxidative stress, which are further aggravated by islet encapsulation, inflammation, and undesirable cell-biomaterial interactions. To identify biomaterials that would not further increase damaging oxidative stress levels and that are also suitable for manufacturing a beta cell encapsulation device, we studied five clinically approved polymers for their effect on oxidative stress and islet (alpha and beta cell) function. We found that 300 poly(ethylene oxide terephthalate) 55/poly(butylene terephthalate) 45 (PEOT/PBT300) was more resistant to breakage and more elastic than other biomaterials, which is important for its immunoprotective function. In addition, it did not induce oxidative stress or reduce viability in the MIN6 beta cell line, and even promoted protective endogenous antioxidant expression over 7 days. Importantly, PEOT/PBT300 is one of the biomaterials we studied that did not interfere with insulin secretion in human islets.

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.

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.

Poly(caprolactone-co-trimethylenecarbonate) urethane acrylate resins for digital light processing of bioresorbable tissue engineering implants.

To exploit the usability of Digital Light Processing (DLP) in regenerative medicine, biodegradable, mechanically customizable and well-defined polyester urethane acrylate resins were synthesized based on poly(caprolactone-co-trimethlenecarbonate). By controlling the monomer ratio, the resultant fabricated constructs showed tunable mechanical properties, degradation and attached hMSC morphologies.

Building Complex Life Through Self-Organization.

Cells are inherently conferred with the ability to self-organize into the tissues and organs comprising the human body. Self-organization can be recapitulated in vitro and recent advances in the organoid field are just one example of how we can generate small functioning elements of organs. Tissue engineers can benefit from the power of self-organization and should consider how they can harness and enhance the process with their constructs. For example, aggregates of stem cells and tissue-specific cells benefit from the input of carefully selected biomolecules to guide their differentiation toward a mature phenotype. This can be further enhanced by the use of technologies to provide a physiological microenvironment for self-organization, enhance the size of the constructs, and enable the long-term culture of self-organized structures. Of importance, conducting self-organization should be limited to fine-tuning and should avoid over-engineering that could counteract the power of inherent cellular self-organization. Impact Statement Self-organization is a powerful innate feature of cells that can be fine-tuned but not over-engineered to create new tissues and organs.

Oxygen and nutrient delivery in tissue engineering: Approaches to graft vascularization.

The field of tissue engineering is making great strides in developing replacement tissue grafts for clinical use, marked by the rapid development of novel biomaterials, their improved integration with cells, better-directed growth and differentiation of cells, and improved three-dimensional tissue mass culturing. One major obstacle that remains, however, is the lack of graft vascularization, which in turn renders many grafts to fail upon clinical application. With that, graft vascularization has turned into one of the holy grails of tissue engineering, and for the majority of tissues, it will be imperative to achieve adequate vascularization if tissue graft implantation is to succeed. Many different approaches have been developed to induce or augment graft vascularization, both in vitro and in vivo. In this review, we highlight the importance of vascularization in tissue engineering and outline various approaches inspired by both biology and engineering to achieve and augment graft vascularization.

Soft-molecular imprinted electrospun scaffolds to mimic specific biological tissues.

The fabrication of bioactive scaffolds able to mimic the in vivo cellular microenvironment is a challenge for regenerative medicine. The creation of sites for the selective binding of specific endogenous proteins represents an attractive strategy to fabricate scaffolds able to elicit specific cell response. Here, electrospinning (ESP) and soft-molecular imprinting (soft-MI) techniques were combined to fabricate a soft-molecular imprinted electrospun bioactive scaffold (SMIES) for tissue regeneration. Scaffolds functionalized using different proteins and growth factors (GFs) arranged onto the surface were designed, fabricated and validated with different polyesters, demonstrating the versatility of the developed approach. The scaffolds bound selectively each of the different proteins used, indicating that the soft-MI method allowed fabricating high affinity binding sites on ESP fibers compared to non-imprinted ones. The imprinting of ESP fibers with several GFs resulted in a significant effect on cell behavior. FGF-2 imprinted SMIES promoted cell proliferation and metabolic activity. BMP-2 and TGF-ß3 imprinted SMIES promoted cellular differentiation. These scaffolds hold the potential to be used in a cell-free approach to steer endogenous tissue regeneration in several regenerative medicine applications.

Redox regulation in regenerative medicine and tissue engineering: The paradox of oxygen.

One of the biggest challenges in tissue engineering and regenerative medicine is to incorporate a functioning vasculature to overcome the consequences of a lack of oxygen and nutrients in the tissue construct. Otherwise, decreased oxygen tension leads to incomplete metabolism and the formation of the so-called reactive oxygen species (ROS). Cells have many endogenous antioxidant systems to ensure a balance between ROS and antioxidants, but if this balance is disrupted by factors such as high levels of ROS due to long-term hypoxia, there will be tissue damage and dysfunction. Current attempts to solve the oxygen problem in the field rarely take into account the importance of the redox balance and are instead centred on releasing or generating oxygen. The first problem with this approach is that although oxygen is necessary for life, it is paradoxically also a highly toxic molecule. Furthermore, although some oxygen-generating biomaterials produce oxygen, they also generate hydrogen peroxide, a ROS, as an intermediate product. In this review, we discuss why it would be a superior strategy to supplement oxygen delivery with molecules to safeguard the important redox balance. Redox sensor proteins that can stimulate the anaerobic metabolism, angiogenesis, and enhancement of endogenous antioxidant systems are discussed as promising targets. We propose that redox regulating biomaterials have the potential to tackle some of the challenges related to angiogenesis and that the knowledge in this review will help scientists in tissue engineering and regenerative medicine realize this aim.

Blastocyst-like structures generated solely from stem cells.

The blastocyst (the early mammalian embryo) forms all embryonic and extra-embryonic tissues, including the placenta. It consists of a spherical thin-walled layer, known as the trophectoderm, that surrounds a fluid-filled cavity sheltering the embryonic cells 1 . From mouse blastocysts, it is possible to derive both trophoblast 2 and embryonic stem-cell lines 3 , which are in vitro analogues of the trophectoderm and embryonic compartments, respectively. Here we report that trophoblast and embryonic stem cells cooperate in vitro to form structures that morphologically and transcriptionally resemble embryonic day 3.5 blastocysts, termed blastoids. Like blastocysts, blastoids form from inductive signals that originate from the inner embryonic cells and drive the development of the outer trophectoderm. The nature and function of these signals have been largely unexplored. Genetically and physically uncoupling the embryonic and trophectoderm compartments, along with single-cell transcriptomics, reveals the extensive inventory of embryonic inductions. We specifically show that the embryonic cells maintain trophoblast proliferation and self-renewal, while fine-tuning trophoblast epithelial morphogenesis in part via a BMP4/Nodal-KLF6 axis. Although blastoids do not support the development of bona fide embryos, we demonstrate that embryonic inductions are crucial to form a trophectoderm state that robustly implants and triggers decidualization in utero. Thus, at this stage, the nascent embryo fuels trophectoderm development and implantation.

An antibody based approach for multi-coloring osteogenic and chondrogenic proteins in tissue engineered constructs.

When tissue engineering strategies rely on the combination of three-dimensional (3D) polymeric or ceramic scaffolds with cells to culture implantable tissue constructs in vitro, it is desirable to monitor tissue growth and cell fate to be able to more rationally predict the quality and success of the construct upon implantation. Such a 3D construct is often referred to as a 'black-box' since the properties of the scaffolds material limit the applicability of most imaging modalities to assess important construct parameters. These parameters include the number of cells, the amount and type of tissue formed and the distribution of cells and tissue throughout the construct. Immunolabeling enables the spatial and temporal identification of multiple tissue types within one scaffold without the need to sacrifice the construct. In this report, we concisely review the applicability of antibodies (Abs) and their conjugation chemistries in tissue engineered constructs. With some preliminary experiments, we show an efficient conjugation strategy to couple extracellular matrix Abs to fluorophores. The conjugated probes proved to be effective in determining the presence of collagen type I and type II on electrospun and additive manufactured 3D scaffolds seeded with adult human bone marrow derived mesenchymal stromal cells. The conjugation chemistry applied in our proof of concept study is expected to be applicable in the coupling of any other fluorophore or particle to the Abs. This could ultimately lead to a library of probes to permit high-contrast imaging by several imaging modalities.

Ectopic bone formation by aggregated mesenchymal stem cells from bone marrow and adipose tissue: A comparative study.

Tissue engineered constructs (TECs) based on spheroids of bone marrow mesenchymal stromal cells (BM-MSCs) combined with calcium phosphate microparticles and enveloped in a platelet-rich plasma hydrogel showed that aggregation of MSCs improves their ectopic bone formation potential. The stromal vascular fraction (SVF) and adipose-derived MSCs (ASCs) have been recognized as an interesting MSC source for bone tissue engineering, but their ectopic bone formation is limited. We investigated whether aggregation of ASCs could similarly improve ectopic bone formation by ASCs and SVF cells. The formation of aggregates with BM-MSCs, ASCs and SVF cells was carried out and gene expression was analysed for osteogenic, chondrogenic and vasculogenic genes in vitro. Ectopic bone formation was evaluated after implantation of TECs in immunodeficient mice with six conditions: TECs with ASCs, TECs with BM-MSC, TECs with SVF cells (with and without rhBMP2), no cells and no cells with rhBMP2. BM-MSCs showed consistent compact spheroid formation, ASCs to a lesser extent and SVF showed poor spheroid formation. Aggregation of ASCs induced a significant upregulation of the expression of osteogenic markers like alkaline phosphatase and collagen type I, as compared with un-aggregated ASCs. In vivo, ASC and SVF cells both generated ectopic bone in the absence of added morphogenetic proteins. The highest incidence of bone formation was seen with BM-MSCs (7/9) followed by SVF + rhBMP2 (4/9) and no cells + rhBMP2 (2/9). Aggregation can improve ectopic bone tissue formation by adipose-derived cells, but is less efficient than rhBMP2. A combination of both factors should now be tested to investigate an additive effect.

The Components of Bone and What They Can Teach Us about Regeneration.

The problem of bone regeneration has engaged both physicians and scientists since the beginning of medicine. Not only can bone heal itself following most injuries, but when it does, the regenerated tissue is often indistinguishable from healthy bone. Problems arise, however, when bone does not heal properly, or when new tissue is needed, such as when two vertebrae are required to fuse to stabilize adjacent spine segments. Despite centuries of research, such procedures still require improved therapeutic methods to be devised. Autologous bone harvesting and grafting is currently still the accepted benchmark, despite drawbacks for clinicians and patients that include limited amounts, donor site morbidity, and variable quality. The necessity for an alternative to this "gold standard" has given rise to a bone-graft and substitute industry, with its central conundrum: what is the best way to regenerate bone? In this review, we dissect bone anatomy to summarize our current understanding of its constituents. We then look at how various components have been employed to improve bone regeneration. Evolving strategies for bone regeneration are then considered.