Background. Post-traumatic chondral and osteochondral lesions can be treated with autologous chondrocyte implantation (ACI), but the high cost of autologous cell expansion under strict Good Manufacturing Practice (GMP) regulations limits patient access. Stem cell-based advanced therapy medicinal products (ATMPs) offer more cost-effective alternatives, with human induced pluripotent stem cells (iPSC) showing great promise due to their expandability, low immunogenicity, commercialization potential, and fewer ethical concerns. Aim. To develop a protocol to direct iPSC through a mesenchymal stage into chondroprogenitors (iCHOp), resembling autologous chondroprogenitor cells used in ACI. Methods. The derived chondroprogenitor cells were expanded in monolayer and in 3-dimensional (3D) cultures and subsequently analyzed using transcriptomic profiling via RNA sequencing and reverse transcription quantitative polymerase chain reaction and compared with ACI chondrocytes. Results. Transcriptomic profiling confirmed successful differentiation, with iCHOp showing 83% similarity to ACI chondrocytes. Further 3D culture maturation led to upregulation of chondrogenesis-related genes and activation of cartilage-specific pathways. Histological analysis confirmed extracellular matrix production, including proteoglycans, collagen, and versican. Furthermore, the protocol's reproducibility was demonstrated using 3 distinct iPSC lines, successfully expanded in both serum-containing and defined serum-free media. Conclusion. Our optimized approach yields iCHOp with phenotypes closely matching ACI chondrocytes, offering a solid foundation for further development and potential clinical applications in cartilage repair.

Stem cell therapy for osteoarthritis: Functional cartilage regeneration using 3d bioprinting technology Osteoarthritis presents a significant societal and economic burden. Stina Simonsson from the University of Gothenburg explains how EU-funded projects are using 3D bioprinting to create functional cartilage for OA treatment. Osteoarthritis (OA) is a widespread condition that affects millions of people globally. As the population ages and the obesity epidemic continues, the incidence of OA is expected to increase. 3D bioprinting and stem cell therapies are heralded as the future of medical treatments for such conditions. OA affects a significant portion of the Swedish population. Estimates suggest that about 7-8% of the population is diagnosed with OA, which translates to roughly 700,000 to 800,000 individuals. The condition is more common among older adults, with a higher prevalence observed in those over the age of 65.

Purpose (the aim of the study): Primary osteoarthritis is a severe, painful and degenerative joint disease with no disease modifying treatment available. Stem cells and biomaterials provide a potential platform for development of new cell based Advanced Medical Therapy Products (ATMP). Human induced pluripotent stem cells (hiPSC) hold promise in the field of regenerative medicine as they show high expandability, do not engender immune reactions and allow easy commercialization and standardization as well as eliminate any ethical concerns.

Autologous fat grafting is hampered by unpredictable outcomes due to high tissue resorption. Hydrogels based on enzymatically pretreated tunicate nanocellulose (ETC) and alginate (ALG) are biocompatible, safe, and present physiochemical properties capable of promoting cell survival. Here, we compared in situ and ex situ crosslinking of ETC/ALG hydrogels combined with lipoaspirate human adipose tissue (LAT) to generate an injectable formulation capable of retaining dimensional stability in vivo. We performed in situ crosslinking using two different approaches; inducing Ca2+ release from CaCO3 microparticles (CMPs) and physiologically available Ca2+ in vivo. Additionally, we generated ex situ-crosslinked, 3D-bioprinted hydrogel-fat grafts. We found that in vitro optimization generated a CMP-crosslinking system with comparable stiffness to ex situ-crosslinked gels. Comparison of outcomes following in vivo injection of each respective crosslinked hydrogel revealed that after 30 days, in situ crosslinking generated fat grafts with less shape retention than 3D-bioprinted constructs that had undergone ex situ crosslinking. However, CMP addition improved fat-cell distribution and cell survival relative to grafts dependent on physiological Ca2+ alone. These findings suggested that in situ crosslinking using CMP might promote the dimensional stability of injectable fat-hydrogel grafts, although 3D bioprinting with ex situ crosslinking more effectively ensured proper shape stability in vivo.

Articular cartilage degeneration poses a significant public health challenge; techniques such as 3D bioprinting are being explored for its regeneration in vitro. Gelatin-based hydrogels represent one of the most promising biopolymers used in cartilage tissue engineering, especially for its collagen composition and tunable mechanical properties. However, there are no standard protocols that define process parameters such as the crosslinking method to apply. To this aim, a reproducible study was conducted for exploring the influence of different crosslinking methods on 3D bioprinted gelatin structures. This study assessed mechanical properties and cell viability in relation to various crosslinking techniques, revealing promising results particularly for dual (photo + ionic) crosslinking methods, which achieved high cell viability and tunable stiffness. These findings offer new insights into the effects of crosslinking methods on 3D bioprinted gelatin for cartilage applications. For example, ionic and photo-crosslinking methods provide softer materials, with photo-crosslinking supporting cell stretching and diffusion, while ionic crosslinking preserves a spherical stem cell morphology. On the other hand, dual crosslinking provides a stiffer, optimized solution for creating stable cartilage-like constructs. The results of this study offer a new perspective on the standardization of gelatin for cartilage bioprinting, bridging the gap between research and clinical applications.

Establishing a vascular network in biofabricated tissue grafts is essential for ensuring graft survival. Such networks are dependent on the ability of the scaffold material to facilitate endothelial cell adhesion; however, the clinical translation potential of tissue-engineered scaffolds is hindered by the lack of available autologous sources of vascular cells. Here, we present a novel approach to achieving autologous endothelialisation in nanocellulose-based scaffolds by using adipose tissue-derived vascular cells on nanocellulose-based scaffolds. We used sodium periodate-mediated bioconjugation to covalently bind laminin to the scaffold surface and isolated the stromal vascular fraction and endothelial progenitor cells (EPCs; CD31 + CD45 − ) from human lipoaspirate. Additionally, we assessed the adhesive capacity of scaffold bioconjugation in vitro using both adipose tissue-derived cell populations and human umbilical vein endothelial cells. The results showed that the bioconjugated scaffold exhibited remarkably higher cell viability and scaffold surface coverage by adhesion regardless of cell type, whereas control groups comprising cells on non-bioconjugated scaffolds exhibited minimal cell adhesion across all cell types. Furthermore, on culture day 3, EPCs seeded on laminin-bioconjugated scaffolds showed positive immunofluorescence staining for the endothelial markers CD31 and CD34, suggesting that the scaffolds promoted progenitor differentiation into mature endothelial cells. These findings present a possible strategy for generating autologous vasculature and thereby increase the clinical relevance of 3D-bioprinted nanocellulose-based constructs.

Abstract Objectives Patients with urethral stricture due to any type of trauma, hypospadias or gender dysphoria suffer immensely from impaired capacity to urinate and are in need of a new functional urethra. Tissue engineering with decellularization of a donated organ recellularized with cells from the recipient patient has emerged as a promising alternative of advanced therapy medicinal products. The aim of this pilot study was to develop an ovine model of urethral transplantation and to produce an individualized urethra graft to show proof of function in vivo. Methods Donated urethras from ram abattoir waste were decellularized and further recellularized with autologous buccal mucosa epithelial cells excised from the recipient ram and expanded in vitro. The individualized urethral grafts were implanted by reconstructive surgery in rams replacing 2.5 ± 0.5 cm of the native penile urethra. Results After surgery optimization, three ram had the tissue engineered urethra implanted for one month and two out of three showed a partially regenerated epithelium. Conclusions Further adjustments of the model are needed to achieve a satisfactory proof-of-concept; however, we interpret these findings as a proof of principle and a possible path to develop a functional tissue engineered urethral graft with de- and recellularization and regeneration in vivo after transplantation.

Knee osteoarthritis is the most common joint disease. It causes pain and suffering for affected patients and is the source of major economic costs for healthcare systems. Despite ongoing research, there is a lack of knowledge regarding disease mechanisms, biomarkers, and possible cures. Current treatments do not fulfill patients’ long-term needs, and it often requires invasive surgical procedures with subsequent long periods of rehabilitation. Researchers and companies worldwide are working to find a suitable cell source to engineer or regenerate a functional and healthy articular cartilage tissue to implant in the damaged area. Potential cell sources to accomplish this goal include embryonic stem cells, mesenchymal stem cells, or induced pluripotent stem cells. The differentiation of stem cells into different tissue types is complex, and a suitable concentration range of specific growth factors is vital. The cellular microenvironment during early embryonic development provides crucial information regarding concentrations of signaling molecules and morphogen gradients as these are essential inducers for tissue development. Thus, morphogen gradients implemented in developmental protocols aimed to engineer functional cartilage tissue can potentially generate cells comparable to those within native cartilage. In this review, we have summarized the problems with current treatments, potential cell sources for cell therapy, reviewed the progress of new treatments within the regenerative cartilage field, and highlighted the importance of cell quality, characterization assays, and chemically defined protocols.

OBJECTIVE:Large cartilage defects and osteoarthritis (OA) cause cartilage loss and remain a therapeutic challenge. Three-dimensional (3D) bioprinting with autologous cells using a computer-aided design (CAD) model generated from 3D imaging has the potential to reconstruct patient-specific features that match an articular joint lesion.DESIGN:To scan a human OA tibial plateau with a cartilage defect, retrieved after total knee arthroplasty, following clinical imaging techniques were used: (1) computed tomography (CT), (2) magnetic resonance imaging (MRI), and (3) a 3D scanner. From such a scan, a CAD file was obtained to generate G-code to control 3D bioprinting in situ directly into the tibial plateau lesion.RESULTS:Highest resolution was obtained using the 3D scanner (2.77 times more points/mm2 than CT), and of the 3 devices tested, only the 3D scanner was able to detect the actual OA defect area. Human chondrocytes included in 3D bioprinted constructs produced extracellular matrix and formed cartilage tissue fragments after 2 weeks of differentiation and high levels of a mature splice version of collagen type II (Col IIA type B), characteristic of native articular cartilage and aggrecan (ACAN). Chondrocytes had a mean viability of 81% in prints after day 5 of differentiation toward cartilage and similar viability was detected in control 3D pellet differentiation of chondrocytes (mean viability 72%).CONCLUSION:Articular cartilage can be formed in 3D bioprints. Thus, this 3D bioprinting system with chondrocytes simulating a patient-specific 3D model provides an attractive strategy for future treatments of cartilage defects or early OA.

Objective Large cartilage defects and osteoarthritis (OA) cause cartilage loss and remain a therapeutic challenge. Three-dimensional (3D) bioprinting with autologous cells using a computer-aided design (CAD) model generated from 3D imaging has the potential to reconstruct patient-specific features that match an articular joint lesion. Design To scan a human OA tibial plateau with a cartilage defect, retrieved after total knee arthroplasty, following clinical imaging techniques were used: (1) computed tomography (CT), (2) magnetic resonance imaging (MRI), and (3) a 3D scanner. From such a scan, a CAD file was obtained to generate G-code to control 3D bioprinting in situ directly into the tibial plateau lesion. Results Highest resolution was obtained using the 3D scanner (2.77 times more points/mm 2 than CT), and of the 3 devices tested, only the 3D scanner was able to detect the actual OA defect area. Human chondrocytes included in 3D bioprinted constructs produced extracellular matrix and formed cartilage tissue fragments after 2 weeks of differentiation and high levels of a mature splice version of collagen type II (Col IIA type B), characteristic of native articular cartilage and aggrecan (ACAN). Chondrocytes had a mean viability of 81% in prints after day 5 of differentiation toward cartilage and similar viability was detected in control 3D pellet differentiation of chondrocytes (mean viability 72%). Conclusion Articular cartilage can be formed in 3D bioprints. Thus, this 3D bioprinting system with chondrocytes simulating a patient-specific 3D model provides an attractive strategy for future treatments of cartilage defects or early OA. Keywords osteoarthritis , 3D bioprinting , chondrocytes , 3D CAD model , cartilage

Organs and tissues, such as cartilage and limbs, are formed during development through an orchestration of growth factors that function as morphogens. Examples of growth factors include growth differentiation factor 5 (GDF5) and transforming growth factors beta 1 and 3 (TGFβ-1 and TGFβ-3) which can specify creation of more than one cell type after forming a concentration gradient in vivo. Here, we studied the impact of morphogen gradients during differentiation of induced pluripotent stem cells (iPSCs) into the chondrocyte lineage. Cell budding zones, consisting of condensed cell aggregates, were observed only in gradients of GDF5. T-box transcription factor 3 (TBX3) was detected specifically in the budding zones (ranging from 500–1,500 particles/μm2) of nuclei and cell vesicles. A homogenous density of GDF5 of 900 particles/μm2 on a surface induced budding and expression of TBX3 after five days in iPSCs. Therefore, we conclude that a gradient of GDF5, as well as the specific homogenous density of GDF5, support the induction of TBX3 in iPCSs. Moreover, differentiation of iPSCs first on GDF5 gradient or homogenous surfaces for five days and then in a three-dimensional structure for five weeks resulted in pellets that expressed TBX3.

Human pluripotent stem cell-derived hepatocytes (hPSC-HEP) display many properties of mature hepatocytes, including expression of important genes of the drug metabolizing machinery, glycogen storage, and production of multiple serum proteins. To this date, hPSC-HEP do not, however, fully recapitulate the complete functionality of in vivo mature hepatocytes. In this study, we applied versatile bioinformatic algorithms, including functional annotation and pathway enrichment analyses, transcription factor binding-site enrichment, and similarity and correlation analyses, to datasets collected from different stages during hPSC-HEP differentiation and compared these to developmental stages and tissues from fetal and adult human liver. Our results demonstrate a high level of similarity between the in vitro differentiation of hPSC-HEP and in vivo hepatogenesis. Importantly, the transcriptional correlation of hPSC-HEP with adult liver (AL) tissues was higher than with fetal liver (FL) tissues (0.83 and 0.70, respectively). Functional data revealed mature features of hPSC-HEP including cytochrome P450 enzymes activities and albumin secretion. Moreover, hPSC-HEP showed expression of many genes involved in drug absorption, distribution, metabolism, and excretion. Despite the high similarities observed, we identified differences of specific pathways and regulatory players by analyzing the gene expression between hPSC-HEP and AL. These findings will aid future intervention and improvement of in vitro hepatocyte differentiation protocol in order to generate hepatocytes displaying the complete functionality of mature hepatocytes. Finally, on the transcriptional level, our results show stronger correlation and higher similarity of hPSC-HEP to AL than to FL. In addition, potential targets for further functional improvement of hPSC-HEP were also identified.

Cells from elderly individuals can by scientists be reprogrammed back to totipotent or pluripotent cells. The first method that achieved reprogramming to totipotency (and thereby pluripotency) was cloning of animals, for example, creation of cloned frogs or Dolly. We and others identified that the protein Oct4 encoded by Pou5f1 gene was a key player in that reprogramming process. Further, epigenetic modifications in the control region of Pou5f1 needed to be reset for the cloning procedure to succeed. The iPS (induced pluripotent stem) cell technology generates pluripotent cells. These cells are promising candidates for regenerative medicine, because of their capability to multiply and differentiate into any cell of interest. Today, a clinical trial is ongoing using cells derived from iPS cells for macular degenerative disease. The iPS cells are also useful for studying human diseases in a dish and drug-screening purposes. The purpose of this article is to introduce the iPS technology and to present the so-called Yamanaka factors; Oct4, c-Myc, Klf4 and Sox2 in the context of development, generation of iPS cells and in cancer.

Background Today, several hundreds of million people all over the world are suffering from different joint disorders; like osteoarthritis (OA). Further, traumatic cartilage lesions can develop into OA. Recent studies indicate that human-derived induced pluripotent stem cells (iPSCs) can be 3D bioprinted and directed to form cartilage-like tissue, thus offering new approaches to treat cartilage lessions.Nguyen et al. 2017 The advantages of using an established iPSC line are unlimited cell source with regeneration capacity and chondrogenic differentiation potential. Objectives The aim of this study was to improve the generation of cartilage-like tissue when 3D bioprinting of iPSCs by using molecularly modified nanocelullose/alginate bioink to resemble natural environment found in the tissue. Methods In this study the chondrocyte-derived iPSC line “A2B” was used (Borestrom et al. 2014). These cells were bioprinted in combination with a modified nanocellulose and alginate bioink. Constructs obtained after the 3D bioprinting were cultured in chondrogenic medium in order to stimulate chondrogenic differentiation. Cell number and viability inside the prints was studied and histological analyses of the 3D printed constructs were performed. Furthermore, expression of pluripotency and chondrogenic specific genes was assessed by Taqman qPCR before and after differentiation. Results After 3D bioprinting high cell viability was found inside the constructs. 3D printed constructs were positively stained for alcian blue van gieson (AB-vG) staining, showing proteoglycans presence inside the prints (figure 1B). Molecular analyses showed high relative expression levels of the pluripotency-related gene Oct4 before starting the differentiation protocol. Cells inside the constructs express chondrogenic specific genes, such as collagen type 2 and Sox9 after 6 weeks of differentiation. Moreover, 3D printed constructs showed cartilage-resembles (figure 1A). Conclusions The 3D printing of the iPSc and the in vitro generation of cartilage-like tissue was successfully achieved using the modified nanocellulose/alginate bioink. This approach could be use in the future to model OA disease and to perform screenings of different therapeutic compounds. References [1] Nguyen D, et al. Cartilage Tissue Engineering by the 3D Bioprinting of iPS Cells in a Nanocellulose/Alginate Bioink. Sci Rep2017;7(1):658. [2] Borestrom C, et al. Footprint-free human induced pluripotent stem cells from articular cartilage with redifferentiation capacity: a first step toward a clinical-grade cell source. Stem Cells Transl Med2014;3;433–447. Disclosure of Interest None declared

Cartilage lesions can progress into secondary osteoarthritis and cause severe clinical problems in numerous patients. As a prospective treatment of such lesions, human-derived induced pluripotent stem cells (iPSCs) were shown to be 3D bioprinted into cartilage mimics using a nanofibrillated cellulose (NFC) composite bioink when co-printed with irradiated human chondrocytes. Two bioinks were investigated: NFC with alginate (NFC/A) or hyaluronic acid (NFC/HA). Low proliferation and phenotypic changes away from pluripotency were seen in the case of NFC/HA. However, in the case of the 3D-bioprinted NFC/A (60/40, dry weight % ratio) constructs, pluripotency was initially maintained, and after five weeks, hyaline-like cartilaginous tissue with collagen type II expression and lacking tumorigenic Oct4 expression was observed in 3D -bioprinted NFC/A (60/40, dry weight % relation) constructs. Moreover, a marked increase in cell number within the cartilaginous tissue was detected by 2-photon fluorescence microscopy, indicating the importance of high cell densities in the pursuit of achieving good survival after printing. We conclude that NFC/A bioink is suitable for bioprinting iPSCs to support cartilage production in co-cultures with irradiated chondrocytes.

Hepatocytes derived from human pluripotent stem cells (hPSC-HEP) have the potential to replace presently used hepatocyte sources applied in liver disease treatment and models of drug discovery and development. Established hepatocyte differentiation protocols are effective and generate hepatocytes, which recapitulate some key features of their in vivo counterparts. However, generating mature hPSC-HEP remains a challenge. In this study, we applied transcriptomics to investigate the progress of in vitro hepatic differentiation of hPSCs at the developmental stages, definitive endoderm, hepatoblasts, early hPSC-HEP, and mature hPSC-HEP, to identify functional targets that enhance efficient hepatocyte differentiation. Using functional annotation, pathway and protein interaction network analyses, we observed the grouping of differentially expressed genes in specific clusters representing typical developmental stages of hepatic differentiation. In addition, we identified hub proteins and modules that were involved in the cell cycle process at early differentiation stages. We also identified hub proteins that differed in expression levels between hPSC-HEP and the liver tissue controls. Moreover, we identified a module of genes that were expressed at higher levels in the liver tissue samples than in the hPSC-HEP. Considering that hub proteins and modules generally are essential and have important roles in the protein-protein interactions, further investigation of these genes and their regulators may contribute to a better understanding of the differentiation process. This may suggest novel target pathways and molecules for improvement of hPSC-HEP functionality, having the potential to finally bring this technology to a wider use.