A Polymer Canvas with the Stiffness of the Bone Matrix to Study and Control Mesenchymal Stem Cell Response.

Reproducing in vitro the complex multiscale physical features of human tissues creates novel biomedical opportunities and fundamental understanding of cell-environment interfaces and interactions. While stiffness has been recognized as a key driver of cell behavior, systematic studies on the role of stiffness have been limited to values in the KPa-MPa range, significantly below the stiffness of bone. Here, a platform enabling the tuning of the stiffness of a biocompatible polymeric interface up to values characteristic of human bone is reported, which are in the GPa range, by using extremely thin polymer films on glass and cross-linking the films using ultraviolet (UV) light irradiation. It is shown that a higher stiffness is related to better adhesion, proliferation, and osteogenic differentiation, and that it is possible to switch on/off cell attachment and growth by solely tuning the stiffness of the interface, without any surface chemistry or topography modification. Since the stiffness is tuned directly by UV irradiation, this platform is ideal for rapid and simple fabrication of stiffness patterns and gradients, thus representing an innovative tool for combinatorial studies of the synergistic effect of tissue environmental cues on cell behavior, and creates new opportunities for next-generation biosensors, single-cell patterning, and lab-on-a-chip devices.

Human induced mesenchymal stem cells display increased sensitivity to matrix stiffness.

The clinical translation of mesenchymal stem cells (MSCs) is limited by population heterogeneity and inconsistent responses to engineered signals. Specifically, the extent in which MSCs respond to mechanical cues varies significantly across MSC lines. Although induced pluripotent stem cells (iPSCs) have recently emerged as a novel cell source for creating highly homogeneous MSC (iMSC) lines, cellular mechanosensing of iMSCs on engineered materials with defined mechanics is not well understood. Here, we tested the mechanosensing properties of three human iMSC lines derived from iPSCs generated using a fully automated platform. Stiffness-driven changes in morphology were comparable between MSCs and iMSCs cultured atop hydrogels of different stiffness. However, contrary to tissue derived MSCs, no significant changes in iMSC morphology were observed between iMSC lines atop different stiffness hydrogels, demonstrating a consistent response to mechanical signals. Further, stiffness-driven changes in mechanosensitive biomarkers were more pronounced in iMSCs than MSCs, which shows that iMSCs are more adaptive and responsive to mechanical cues than MSCs. This study reports that iMSCs are a promising stem cell source for basic and applied research due to their homogeneity and high sensitivity to engineered mechanical signals.

GMP-Compatible, Xeno-Free Culture of Human Induced Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have been used in therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. To foster commercialization and implementation of stem cells treatments, researchers have recently derived MSCs from human induced pluripotent stem cells (iMSCs). For therapeutic applications, human iMSCs must be produced in xeno-free culture conditions and following procedures that are compatible with the principles of Good Manufacturing Practice.

A biomimetic engineered bone platform for advanced testing of prosthetic implants.

Existing methods for testing prosthetic implants suffer from critical limitations, creating an urgent need for new strategies that facilitate research and development of implants with enhanced osseointegration potential. Herein, we describe a novel, biomimetic, human bone platform for advanced testing of implants in vitro, and demonstrate the scientific validity and predictive value of this approach using an assortment of complementary evaluation methods. We anchored titanium (Ti) and stainless steel (SS) implants into biomimetic scaffolds, seeded with human induced mesenchymal stem cells, to recapitulate the osseointegration process in vitro. We show distinct patterns of gene expression, matrix deposition, and mineralization in response to the two materials, with Ti implants ultimately resulting in stronger integration strength, as seen in other preclinical and clinical studies. Interestingly, RNAseq analysis reveals that the TGF-beta and the FGF2 pathways are overexpressed in response to Ti implants, while the Wnt, BMP, and IGF pathways are overexpressed in response to SS implants. High-resolution imaging shows significantly increased tissue mineralization and calcium deposition at the tissue-implant interface in response to Ti implants, contributing to a twofold increase in pullout strength compared to SS implants. Our technology creates unprecedented research opportunities towards the design of implants and biomaterials that can be personalized, and exhibit enhanced osseointegration potential, with reduced need for animal testing.

Hypothermic and cryogenic preservation of tissue-engineered human bone.

To foster translation and commercialization of tissue-engineered products, preservation methods that do not significantly compromise tissue properties need to be designed and tested. Robust preservation methods will enable the distribution of tissues to third parties for research or transplantation, as well as banking of off-the-shelf products. We recently engineered bone grafts from induced pluripotent stem cells and devised strategies to facilitate a tissue-engineering approach to segmental bone defect therapy. In this study, we tested the effects of two potential preservation methods on the survival, quality, and function of tissue-engineered human bone. Engineered bone grafts were cultured for 5 weeks in an osteogenic environment and then stored in phosphate-buffered saline (PBS) solution at 4 °C or in Synth-a-Freeze™ at -80 °C. After 48 h, samples were warmed up in a water bath at 37 °C, incubated in osteogenic medium, and analyzed 1 and 24 h after revitalization. The results show that while storage in Synth-a-Freeze at -80 °C results in cell death and structural alteration of the extracellular matrix, hypothermic storage in PBS does not significantly affect tissue viability and integrity. This study supports the use of short-term hypothermic storage for preservation and distribution of high-quality tissue-engineered bone grafts for research and future clinical applications.

GMP-compatible and xeno-free cultivation of mesenchymal progenitors derived from human-induced pluripotent stem cells.

BACKGROUND: Human mesenchymal stem cells are a strong candidate for cell therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. Yet, their scarcity, limited expansion potential, and age-associated functional decline restrict the ability to consistently manufacture large numbers of safe and therapeutically effective mesenchymal stem cells for routine clinical applications. To overcome these limitations and advance stem cell treatments using mesenchymal stem cells, researchers have recently derived mesenchymal progenitors from human-induced pluripotent stem cells. Human-induced pluripotent stem cell-derived progenitors resemble adult mesenchymal stem cells in morphology, global gene expression, surface antigen profile, and multi-differentiation potential, but unlike adult mesenchymal stem cells, it can be produced in large numbers for every patient. For therapeutic applications, however, human-induced pluripotent stem cell-derived progenitors must be produced without animal-derived components (xeno-free) and in accordance with Good Manufacturing Practice guidelines. METHODS: In the present study we investigate the effects of expanding mesodermal progenitor cells derived from two human-induced pluripotent stem cell lines in xeno-free medium supplemented with human platelet lysates and in a commercial high-performance Good Manufacturing Practice-compatible medium (Unison Medium). RESULTS: The results show that long-term culture in xeno-free and Good Manufacturing Practice-compatible media somewhat affects the morphology, expansion potential, gene expression, and cytokine profile of human-induced pluripotent stem cell-derived progenitors but supports cell viability and maintenance of a mesenchymal phenotype equally well as medium supplemented with fetal bovine serum. CONCLUSIONS: The findings support the potential to manufacture large numbers of clinical-grade human-induced pluripotent stem cell-derived mesenchymal progenitors for applications in personalized regenerative medicine.

Comparison of Decellularized Cow and Human Bone for Engineering Bone Grafts with Human Induced Pluripotent Stem Cells.

IMPACT STATEMENT: Decellularized tissue matrices are popular as scaffolding materials for tissue engineering application. However, it is unclear whether interspecies differences in tissue parameters influence the quality of tissue grafts that are engineered using human stem cells. In this study, decellularized cow and human bone scaffolds were compared for engineering bone grafts using human induced pluripotent stem cell-derived mesodermal progenitor cells and despite minor differences in architecture and mass composition, both scaffolds equally support cell viability and tissue mineralization. Decellularized cow bone scaffolds therefore represent a suitable and more affordable alternative for engineering human bone grafts for basic and applied research.

Segmental Additive Tissue Engineering.

Segmental bone defects caused by trauma and disease represent a major clinical problem worldwide. Current treatment options are limited and often associated with poor outcomes and severe complications. Bone engineering is a promising alternative solution, but a number of technical challenges must be addressed to allow for effective and reproducible construction of segmental grafts that meet the size and geometrical requirements needed for individual patients and routine clinical applications. It is important to devise engineering strategies and standard operating procedures that make it possible to scale up the size of bone-engineered grafts, minimize process and product variability, and facilitate technology transfer and implementation. To address these issues, we have combined traditional and modular tissue engineering approaches in a strategy referred to as Segmental Additive Tissue Engineering (SATE). To demonstrate this approach, a digital reconstruction of a rabbit femoral defect was partitioned transversally to the longitudinal axis into segments (modules) with discoidal geometry and defined thickness to enable protocol standardization and effective tissue formation in vitro. Bone grafts corresponding to each segment were then engineered using biomimetic scaffolds seeded with human induced pluripotent stem cell-derived mesodermal progenitors (iPSC-MPs) and a novel perfusion bioreactor with universal design. The SATE strategy enables the effective and reproducible engineering of segmental bone grafts for personalized skeletal reconstruction, and will facilitate technology transfer and implementation of a tissue engineering approach to segmental bone defect therapy.

Engineering human bone grafts with new macroporous calcium phosphate cement scaffolds.

Bone engineering opens the possibility to grow large amounts of tissue products by combining patient-specific cells with compliant biomaterials. Decellularized tissue matrices represent suitable biomaterials, but availability, long processing time, excessive cost, and concerns on pathogen transmission have led to the development of biomimetic synthetic alternatives. We recently fabricated calcium phosphate cement (CPC) scaffolds with variable macroporosity using a facile synthesis method with minimal manufacturing steps and demonstrated long-term biocompatibility in vitro. However, there is no knowledge on the potential use of these scaffolds for bone engineering and whether the porosity of the scaffolds affects osteogenic differentiation and tissue formation in vitro. In this study, we explored the bone engineering potential of CPC scaffolds with two different macroporosities using human mesenchymal progenitors derived from induced pluripotent stem cells (iPSC-MP) or isolated from bone marrow (BMSC). Biomimetic decellularized bone scaffolds were used as reference material in all experiments. The results demonstrate that, irrespective of their macroporosity, the CPC scaffolds tested in this study support attachment, viability, and growth of iPSC-MP and BMSC cells similarly to decellularized bone. Importantly, the tested materials sustained differentiation of the cells as evidenced by increased expression of osteogenic markers and formation of a mineralized tissue. In conclusion, the results of this study suggest that the CPC scaffolds fabricated using our method are suitable to engineer bone grafts from different cell sources and could lead to the development of safe and more affordable tissue grafts for reconstructive dentistry and orthopaedics and in vitro models for basic and applied research.

Stem cell-mediated functionalization of titanium implants.

Prosthetic implants are used daily to treat edentulous people and to restore mobility in patients affected by skeletal defects. Titanium (Ti) is the material of choice in prosthetics, because it can form a stable bond with the surrounding bone following implantation-a process known as osseointegration. Yet, full integration of prosthetic implants takes time, and fails in clinical situations characterized by limited bone quantity and/or compromised regenerative capacity, and in at-risk patients. Intense research efforts are thus made to develop new implants that are cost-effective, safe, and suited to every patient in each clinical situation. In this study, we tested the possibility to functionalize Ti implants using stem cells. Human induced pluripotent stem cell-derived mesenchymal progenitor (iPSC-MP) cells were cultured on Ti model disks for 2 weeks in osteogenic conditions. Samples were then treated using four different decellularization methods to wash off the cells and expose the matrix. The functionalized disks were finally sterilized and seeded with fresh human iPSC-MP cells to study the effect of stem cell-mediated surface functionalization on cell behavior. The results show that different decellularization methods produce diverse surface modifications, and that these modifications promote proliferation of human iPSC-MP cells, affect the expression of genes involved in development and differentiation, and stimulate the release of alkaline phosphatase. Cell-mediated functionalization represents an attractive strategy to modify the surface of prosthetic implants with cues of biological relevance, and opens unprecedented possibilities for development of new devices with enhanced therapeutic potential.

Fabrication of macroporous cement scaffolds using PEG particles: In vitro evaluation with induced pluripotent stem cell-derived mesenchymal progenitors.

Calcium phosphate cements (CPCs) have been extensively used in reconstructive dentistry and orthopedics, but it is only recently that CPCs have been combined with stem cells to engineer biological substitutes with enhanced healing potential. In the present study, macroporous CPC scaffolds with defined composition were fabricated using an easily reproduced synthesis method, with minimal fabrication and processing steps. Scaffold pore size and porosity, essential for cell infiltration and tissue ingrowth, were tuned by varying the content and size of polyethylene glycol (PEG) particles, resulting in 9 groups with different architectural features. The scaffolds were characterized for chemical composition, porosity and mechanical properties, then tested in vitro with human mesenchymal progenitors derived from induced pluripotent stem cells (iPSC-MPs). Biomimetic decellularized bone scaffolds were used as reference material in this study. Our manufacturing process resulted in the formation of macroporous monetite scaffolds with no residual traces of PEG. The size and content of PEG particles was found to affect scaffold porosity, and thus mechanical properties. Irrespective of pore size and porosity, the CPC scaffolds fabricated in this study supported adhesion and viability of human iPSC-MPs similarly to decellularized bone scaffolds. However, the architectural features of the scaffolds were found to affect the expression of bone specific genes, suggesting that specific scaffold groups could be more suitable to direct human iPSC-MPs in vitro toward an osteoblastic phenotype. Our simplistic fabrication method allows rapid, inexpensive and reproducible construction of macroporous CPC scaffolds with tunable architecture for potential use in dental and orthopedic applications.

Osteogenic response of human mesenchymal stem cells to well-defined nanoscale topography in vitro.

BACKGROUND: Patterning medical devices at the nanoscale level enables the manipulation of cell behavior and tissue regeneration, with topographic features recognized as playing a significant role in the osseointegration of implantable devices. METHODS: In this study, we assessed the ability of titanium-coated hemisphere-like topographic nanostructures of different sizes (approximately 50, 100, and 200 nm) to influence the morphology, proliferation, and osteogenic differentiation of human mesenchymal stem cells (hMSCs). RESULTS: We found that the proliferation and osteogenic differentiation of hMSCs was influenced by the size of the underlying structures, suggesting that size variations in topographic features at the nanoscale level, independently of chemistry, can be exploited to control hMSC behavior in a size-dependent fashion. CONCLUSION: Our studies demonstrate that colloidal lithography, in combination with coating technologies, can be exploited to investigate the cell response to well defined nanoscale topography and to develop next-generation surfaces that guide tissue regeneration and promote implant integration.

Cultivation of human bone-like tissue from pluripotent stem cell-derived osteogenic progenitors in perfusion bioreactors.

Human pluripotent stem cells represent an unlimited source of skeletal tissue progenitors for studies of bone biology, pathogenesis, and the development of new approaches for bone reconstruction and therapies. In order to construct in vitro models of bone tissue development and to grow functional, clinical-size bone substitutes for transplantation, cell cultivation in three-dimensional environments composed of porous osteoconductive scaffolds and dynamic culture systems-bioreactors-has been studied. Here, we describe a stepwise procedure for the induction of human embryonic and induced pluripotent stem cells (collectively termed PSCs) into mesenchymal-like progenitors, and their subsequent cultivation on decellularized bovine bone scaffolds in perfusion bioreactors, to support the development of viable, stable bone-like tissue in defined geometries.

Make no bones about it: cells could soon be reprogrammed to grow replacement bones?

Recent developments in nuclear reprogramming allow the generation of patient-matched stem cells with broad potential for applications in cell therapies, disease modeling and drug discovery. An increasing body of work is reporting the derivation of lineage-specific progenitors from human-induced pluripotent stem cells (hiPSCs), which could in the near future be used to engineer personalized tissue substitutes, including those for reconstructive therapies of bone. Although the potential clinical impact of such technology is not arguable, significant challenges remain to be addressed before hiPSC-derived progenitors can be employed to engineer bone substitutes of clinical relevance. The most important challenge is indeed the construction of personalized multicellular bone substitutes for the treatment of complex skeletal defects that integrate fast, are immune tolerated and display biofunctionality and long-term safety. As recent studies suggest, the merging of iPSC technology with advanced biomaterials and bioreactor technologies offers a way to generate bone substitutes in a controllable, automated manner with potential to meet the needs for scale-up and requirements for translation into clinical practice. It is only via the use of state-of-the-art cell culture technologies, process automation under GMP-compliant conditions, application of appropriate engineering strategies and compliance with regulatory policies that personalized lab-made bone grafts can start being used to treat human patients.

Modulating the biochemical and biophysical culture environment to enhance osteogenic differentiation and maturation of human pluripotent stem cell-derived mesenchymal progenitors.

Advances in the fields of stem cell biology, biomaterials, and tissue engineering over the last decades have brought the possibility of constructing tissue substitutes with a broad range of applications in regenerative medicine, disease modeling, and drug discovery. Different types of human stem cells have been used, each presenting a unique set of advantages and limitations with regard to the desired research goals. Whereas adult stem cells are at the frontier of research for tissue and organ regeneration, pluripotent stem cells represent a more challenging cell source for clinical translation. However, with their unlimited growth and wide differentiation potential, pluripotent stem cells represent an unprecedented resource for the construction of advanced human tissue models for biological studies and drug discovery. At the heart of these applications lies the challenge to reproducibly expand, differentiate, and organize stem cells into mature, stable tissue structures. In this review, we focus on the derivation of mesenchymal tissue progenitors from human pluripotent stem cells and the control of their osteogenic differentiation and maturation by modulation of the biophysical culture environment. Similarly to enhancing bone development, the described principles can be applied to the construction of other mesenchymal tissues for basic and applicative studies.

Engineering bone tissue substitutes from human induced pluripotent stem cells.

Congenital defects, trauma, and disease can compromise the integrity and functionality of the skeletal system to the extent requiring implantation of bone grafts. Engineering of viable bone substitutes that can be personalized to meet specific clinical needs represents a promising therapeutic alternative. The aim of our study was to evaluate the utility of human-induced pluripotent stem cells (hiPSCs) for bone tissue engineering. We first induced three hiPSC lines with different tissue and reprogramming backgrounds into the mesenchymal lineages and used a combination of differentiation assays, surface antigen profiling, and global gene expression analysis to identify the lines exhibiting strong osteogenic differentiation potential. We then engineered functional bone substitutes by culturing hiPSC-derived mesenchymal progenitors on osteoconductive scaffolds in perfusion bioreactors and confirmed their phenotype stability in a subcutaneous implantation model for 12 wk. Molecular analysis confirmed that the maturation of bone substitutes in perfusion bioreactors results in global repression of cell proliferation and an increased expression of lineage-specific genes. These results pave the way for growing patient-specific bone substitutes for reconstructive treatments of the skeletal system and for constructing qualified experimental models of development and disease.

Human embryonic stem cell-derived mesodermal progenitors display substantially increased tissue formation compared to human mesenchymal stem cells under dynamic culture conditions in a packed bed/column bioreactor.

Bone tissue engineering represents a promising strategy to obviate bone deficiencies, allowing the ex vivo construction of bone substitutes with unprecedented potential in the clinical practice. Considering that in the human body cells are constantly stimulated by chemical and mechanical stimuli, the use of bioreactor is emerging as an essential factor for providing the proper environment for the reproducible and large-scale production of the engineered substitutes. Human mesenchymal stem cells (hMSCs) are experimentally relevant cells but, regardless the encouraging results reported after culture under dynamic conditions in bioreactors, show important limitations for tissue engineering applications, especially considering their limited proliferative potential, loss of functionality following protracted expansion, and decline in cellular fitness associated with aging. On the other hand, we previously demonstrated that human embryonic stem cell-derived mesodermal progenitors (hES-MPs) hold great potential to provide a homogenous and unlimited source of cells for bone engineering applications. Based on prior scientific evidence using different types of stem cells, in the present study we hypothesized that dynamic culture of hES-MPs in a packed bed/column bioreactor had the potential to affect proliferation, expression of genes involved in osteogenic differentiation, and matrix mineralization, therefore resulting in increased bone-like tissue formation. The reported findings suggest that hES-MPs constitute a suitable alternative cell source to hMSCs and hold great potential for the construction of bone substitutes for tissue engineering applications in clinical settings.

The sixth annual translational stem cell research conference of the New York Stem Cell Foundation.

The New York Stem Cell Foundation's "Sixth Annual Translational Stem Cell Research Conference" convened on October 11-12, 2011 at the Rockefeller University in New York City. Over 450 scientists, patient advocates, and stem cell research supporters from 14 countries registered for the conference. In addition to poster and platform presentations, the conference featured panels entitled "Road to the Clinic" and "The Future of Regenerative Medicine."

State of the art in stem cell research: human embryonic stem cells, induced pluripotent stem cells, and transdifferentiation.

Stem cells divide by asymmetric division and display different degrees of potency, or ability to differentiate into various specialized cell types. Owing to their unique regenerative capacity, stem cells have generated great enthusiasm worldwide and represent an invaluable tool with unprecedented potential for biomedical research and therapeutic applications. Stem cells play a central role in the understanding of molecular mechanisms regulating tissue development and regeneration in normal and pathological conditions and open large possibilities for the discovery of innovative pharmaceuticals to treat the most devastating diseases of our time. Not least, their intrinsic characteristics allow the engineering of functional tissues for replacement therapies that promise to revolutionize the medical practice in the near future. In this paper, the authors present the characteristics of pluripotent stem cells and new developments of transdifferentiation technologies and explore some of the biomedical applications that this emerging technology is expected to empower.

Superior osteogenic capacity of human embryonic stem cells adapted to matrix-free growth compared to human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) represent a promising source of cells for bone tissue engineering. However, their low frequencies and limited proliferation restrict their clinical utility. An alternative is the use of human embryonic stem cells (hESCs), but labor-intensive expansion with the need for coating support limits their clinical use. We have previously derived a cell line from hESCs denoted matrix-free growth (MFG)-hESC that are independent of coating support for expansion, and we here compare its osteogenic capacity to that of hMSCs. Microarray analysis of hMSCs and MFG-hESCs revealed differential expression of genes involved in ossification. MFG-hESCs have significantly higher expression of secreted phosphoprotein 1 (SPP1) during osteogenic differentiation, whereas the opposite was true for alkaline phosphatase (ALPL), transforming growth factor, beta 1 (TGFB2), runt-related transcription factor 2 (RUNX2), and forkhead box C1 (FOXC1), as well as the activity of the ALPL enzyme, demonstrating that these two cell types differentiate into the osteogenic lineage using different signaling pathways. von Kossa staining, time-of-flight secondary ion mass spectrometry, and measurement of calcium and phosphate in the extracellular matrix demonstrated a superior ability of the MFG-hESCs to produce a mineralized matrix compared to hMSCs. The superior ability of the MFG-hESCs to form mineralized matrix compared to hMSCs demonstrates that MFG-hESCs are a promising alternative to the use of adult stem cells in future bone regenerative applications.