Adipose Stem Cells in Regenerative Medicine: Looking Forward

Over the last decade, stem cell-based regenerative medicine has progressed to clinical testing and therapeutic applications. The applications range from infusions of autologous and allogeneic stem cells to stem cell-derived products. Adult stem cells from adipose tissue (ASCs) show significant promise in treating autoimmune and neurodegenerative diseases, vascular and metabolic diseases, bone and cartilage regeneration and wound defects. The regenerative capabilities of ASCs in vivo are primarily orchestrated by their secretome of paracrine factors and cell-matrix interactions. More recent developments are focused on creating more complex structures such as 3D organoids, tissue elements and eventually fully functional tissues and organs to replace or repair diseased or damaged tissues. The current and future applications for ASCs in regenerative medicine are discussed here.

Organ on Chip Technology to Model Cancer Growth and Metastasis

Organ on chip (OOC) has emerged as a major technological breakthrough and distinct model system revolutionizing biomedical research and drug discovery by recapitulating the crucial structural and functional complexity of human organs in vitro. OOC are rapidly emerging as powerful tools for oncology research. Indeed, Cancer on chip (COC) can ideally reproduce certain key aspects of the tumor microenvironment (TME), such as biochemical gradients and niche factors, dynamic cell–cell and cell–matrix interactions, and complex tissue structures composed of tumor and stromal cells. Here, we review the state of the art in COC models with a focus on the microphysiological systems that host multicellular 3D tissue engineering models and can help elucidate the complex biology of TME and cancer growth and progression. Finally, some examples of microengineered tumor models integrated with multi-organ microdevices to study disease progression in different tissues will be presented.

3D Bioprinting of Gelatin–Xanthan Gum Composite Hydrogels for Growth of Human Skin Cells

In recent years, bioprinting has attracted much attention as a potential tool for generating complex 3D biological constructs capable of mimicking the native tissue microenvironment and promoting physiologically relevant cell–cell and cell–matrix interactions. The aim of the present study was to develop a crosslinked 3D printable hydrogel based on biocompatible natural polymers, gelatin and xanthan gum at different percentages to be used both as a scaffold for cell growth and as a wound dressing. The CellInk Inkredible 3D printer was used for the 3D printing of hydrogels, and a glutaraldehyde solution was tested for the crosslinking process. We were able to obtain two kinds of printable hydrogels with different porosity, swelling and degradation time. Subsequently, the printed hydrogels were characterized from the point of view of biocompatibility. Our results showed that gelatin/xanthan-gum bioprinted hydrogels were biocompatible materials, as they allowed both human keratinocyte and fibroblast in vitro growth for 14 days. These two bioprintable hydrogels could be also used as a helpful dressing material.

Exploiting maleimide-functionalized hyaluronan hydrogels to test cellular responses to physical and biochemical stimuli

Current in vitro 3D models of liver tissue have been limited by the inability to study the effects of specific extracellular matrix (ECM) components on cell phenotypes. This is in part due to limitations in the availability of chemical modifications appropriate for this purpose. For example, hyaluronic acid (HA), which is a natural ECM component within the liver, lacks key ECM motifs (e.g., RGD peptides) that support cell adhesion. However, the addition of maleimide (Mal) groups to HA could facilitate the conjugation of ECM biomimetic peptides with thiol-containing end groups. In this study, we characterized a new crosslinkable hydrogel (i.e., HA-Mal) that yielded a simplified ECM-mimicking microenvironment supportive of 3D liver cell culture. We then performed a series of experiments to assess the impact of physical and biochemical signaling in the form of RGD peptide incorporation and TGF- ß supplementation, respectively, on hepatic functionality. Hepatic stellate cells (i.e., LX-2) exhibited increased cell-matrix interactions in the form of cell spreading and elongation within HA-Mal matrices containing RGD peptides, enabling physical adhesions, whereas hepatocyte-like cells (HepG2) had reduced albumin and urea production. We further exposed the encapsulated cells to soluble TGF-ß to elicit a fibrosis-like state. In the presence of TGF-ß biochemical signals, LX-2 cells became activated and HepG2 functionality significantly decreased in both RGD-containing and RGD-free hydrogels. Altogether, in this study we have developed a hydrogel biomaterial platform that allows for discrete manipulation of specific ECM motifs within the hydrogel to better understand the roles of cell-matrix interactions on cell phenotype and overall liver functionality.

The role of cell-matrix interactions in connective tissue mechanics

Living tissue is able to withstand large stresses in everyday life, yet it also actively adapts to dynamic loads. This remarkable mechanical behaviour emerges from the interplay between living cells and their non-living extracellular environment. Here we review recent insights into the biophysical mechanisms involved in the reciprocal interplay between cells and the extracellular matrix and how this interplay determines tissue mechanics, with a focus on connective tissues. We first describe the roles of the main macromolecular components of the extracellular matrix in regards to tissue mechanics. We then proceed to highlight the main routes via which cells sense and respond to their biochemical and mechanical extracellular environment. Next we introduce the three main routes via which cells can modify their extracellular environment: exertion of contractile forces, secretion and deposition of matrix components, and matrix degradation. Finally we discuss how recent insights in the mechanobiology of cell-matrix interactions are furthering our understanding of the pathophysiology of connective tissue diseases and cancer, and facilitating the design of novel strategies for tissue engineering.

Regional-specific meniscal extracellular matrix hydrogels and their effects on cell-matrix interactions of fibrochondrocytes

Decellularized meniscal extracellular matrix (ECM) material holds great potential for meniscus repair and regeneration. Particularly, injectable ECM hydrogel is highly desirable for the minimally invasive treatment of irregularly shaped defects. Although regional-specific variations of the meniscus are well documented, no ECM hydrogel has been reported to simulate zonally specific microenvironments of the native meniscus. To fill the gap, different (outer, middle, and inner) zones of porcine menisci were separately decellularized. Then the regionally decellularized meniscal ECMs were solubilized by pepsin digestion, neutralized, and then form injectable hydrogels. The hydrogels were characterized in gelation behaviors and mechanical properties and seeded with bovine fibrochondrocytes to evaluate the regionally biochemical effects on the cell-matrix interactions. Our results showed that the decellularized inner meniscal ECM (IM) contained the greatest glycosaminoglycan (GAG) content and the least collagen content compared with the decellularized outer meniscal ECM (OM) and middle meniscal ECM (MM). The IM hydrogel showed lower compressive strength than the OM hydrogel. When encapsulated with fibrochondrocytes, the IM hydrogel accumulated more GAG, contracted to a greater extent and reached higher compressive strength than that of the OM hydrogel at 28 days. Our findings demonstrate that the regionally specific meniscal ECMs present biochemical variation and show various effects on the cell behaviors, thus providing information on how meniscal ECM hydrogels may be utilized to reconstruct the microenvironments of the native meniscus.

Hydrogel-Based 3D Culture Model for Down Syndrome Associated Transient Myeloproliferative Disorder Using Customized Induced Pluripotent Stem Cells

The generation of hematopoietic stem and progenitor cells (HSPCs) from induced pluripotent stem cells (iPSCs) provides an extraordinary tool for hematological disease modeling of rare disorders such as Down syndrome (DS) associated transient myeloproliferative disorder (TMD). TMD is a preleukemic condition observed in 10-20% of children with trisomy 21 possessing the pathognomonic mutation in the transcription factor GATA1. Hematopoiesis in the bone marrow (BM) is affected by cell-cell and cell-matrix interactions. The current methods for iPSC differentiation into HSPCs utilize either 2-dimensional (2D) monolayer of mouse stromal cells or animal tissue derived extracellular matrices. Generation of a 3-dimensional (3D) culture environment attempts to facilitate both cell-cell and cell-matrix interactions during iPSC differentiation. This study reports the development of a 3D culture system for hematopoietic differentiation of iPSCs to model TMD. iPSC colonies were encapsulated in 3D polyethylene glycol (PEG) based hydrogels containing synthetic integrin binding peptide (GRGDSPC) and enzymatically degradable peptide (GGPQGIWGQGKG) (Fig. 1A) and cultured in maintenance medium (mTeSR1™, Stem Cell Technology) without feeder cells. There were notable morphological differences between the 3D encapsulated and 2D cultured iPSC colonies (Fig. 1B). The 3D encapsulation did not have an adverse effect on the viability of the iPSC colonies evaluated by in situ staining with viability dye (Fig. 1C). The 3D encapsulated colonies were more compact with a spheroid morphology in PEG whereas colonies in 2D were more flattened (Fig. 1D). The pluripotency of the 3D encapsulated iPSCs was confirmed alkaline phosphatase staining (purple colonies) and by the presence of >96% population expressing pluripotency markers, Tra-1-60 and SSEA-4 (Fig. 1E). To test the efficiency of the 3D model system to generate HSPCs, the encapsulated iPSCs were subjected to hematopoietic differentiation using STEMdiff Hematopoietic Kit. Following differentiation, immunophenotype analysis of single cells by flow cytometry revealed a 1.7-fold higher CD34+CD45+CD38-CD45RA- cell percentage in 3D hydrogels compared to 2D. Further delineation of sub-populations in HSPC compartment from 2D and 3D hydrogel revealed a 1.9-fold and 2.1-fold higher population of early HSPCs and multipotent progenitors (MPPs) in 3D compared to 2D respectively (Fig 1F, *P<0.05). In colony forming unit (CFU) assay, the 3D generated HSPCs gave rise to a 2.0-fold higher number of CFU-GEMM (granulocyte, erythrocyte, monocyte, megakaryocyte) colonies compared to 2D, with 2.0-fold decreased number of BFU-E (erythroid) colonies and a similar number of CFU-GM (granulocyte, macrophage) colonies (Fig. 1G). Thus, the low modulus synthetic matrix promoted hematopoietic differentiation producing higher percentage of early HSPCs as compared to the 2D culture system. We used this 3D system to model TMD by utilizing isogenic iPSCs with disomy 21 (D21), trisomy 21 (T21), and trisomy 21 bearing pathologic mutation in GATA1 (T21-G1). The megakaryoid population in the HSPCs generated by hematopoietic differentiation of 3D encapsulated iPSCs was characterized by the percentage of CD34+CD41+ population within the total CD41+ population, myeloid population as CD18+CD45+ and erythroid population as CD71+CD235+. T21 HSPCs showed increased erythroid and megakaryoid populations as compared to isogenic D21, consistent with the role of trisomy 21 in perturbing hematopoiesis. T21-G1 had elevated megakaryoid (93±6% vs 71±1%,) and myeloid (32±16% vs 8±4%) populations with reduced erythroid (27±12% vs 79±6%) population as compared to T21 HSPCs implicating GATA1s in altered hematopoiesis (Fig. 1H). T21-G1 HSPCs only produced CFU-GM colonies as compared to a high number of CFU-GEMM and BFU-E in T21 and D21 HSPCs (Fig. 1I). The expression of GATA1s in T21-G1 megakaryoid population was confirmed (Fig. 1J). The immunophenotype marker analysis of T21-G1 megakaryoid blasts showed expression of megakaryoid/erythroid antigens (CD41, CD61, CD42b, CD71) along with myeloid markers (CD11b, CD33, CD13) and increased expression of CD56 and CD117 consistent with TMD patients (Fig. 1K). In conclusion, our cost-effective tunable 3D hydrogel system promoted hematopoietic differentiation of iPSCs and generated TMD model mimicking the salient features of the disease. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Gopalakrishnapillai: Geron: Research Funding.

Production of Cell Enclosing Silk Derivative Microsphere in Uniform Size Distribution Through Coaxial Microfluidic Device and Horseradish Crosslinking Reaction

BackgroundSilk fibroin (SF) as a natural polymer holds great potential in biomedical research because of its biocompatibility, easy processing, high toughness, and strength. However, slow gelation time has narrowed its applications, specifically in cell-laden microparticles that are versatile structures for tissue engineering due to their unique features. In addition, most crosslinking methods used to decrease gelation time did not occur in a mid-condition. Methods This study aimed to use modified SF with phenol conjugation to accelerate crosslinking mediated via horseradish peroxidase (HRP)/ hydrogen peroxide (H2O2) in a co-flow high-throughput microfluidic device for the ultimate goal of cell-laden silk fibroin-phenol (SF-Ph) microparticles formation. The physical and biochemical properties of fabricated cell-laden SF-Ph were evaluated to reveal its potential for tissue engineering.ResultsThe monodisperse microparticles in shape and size were formed in various diameters changing from 300 to 80 µm by altering oil phase velocity from SF-Ph substrate. More than 90% cell viability and three times cells upregulation of mitochondrial activity of enclosed-cells in microparticles with 150 ± 32 µm diameters revealed that these structures were suitable subcultures produced through a mild process based on morphological and MTT assays. It was noticed that cells approximately cover the microparticles until the 15th day. ConclusionSpherical micro-tissue formation in microparticles, resulting from cell growth promoted by cell-cell and cell-matrix interactions, adds significant weight to this method's applications.