Impact In Regenerative Medicine Research Articles

Cholangiocyte Organoids in Liver Transplantation; a Comprehensive Review.

Liver transplantation is the only curative option for many liver diseases that end up in liver failure, and cholangiopathy remains a challenging complication post-liver transplant, associated with significant morbidity and potential graft loss. The low availability of organs and high demand for transplantation motivate scientists to find novel interventions. Organoids, as three-dimensional cell cultures derived from adult cells or induced pluripotent cells, may help to address this problem. Different types of organoids have been described, from which cholangiocyte organoids offer a high level of versatility and plasticity for a deeper study of liver disease mechanisms. Cholangiocytes can be obtained from different segments of the biliary tree and have shown a remarkable capacity to adapt to new environments, presenting an effective system for studying cholangiopathies. Studies using cholangiocyte organoids show promising results for disease modeling, where organoids offer fundamental features to recapitulate the complexities of tissues in vitro and uncover fundamental pathological pathways to potentially reveal therapeutic strategies for personalized medicine. Organoids could hold the potential for regeneration of injured livers, representing tools of clinical impact in regenerative medicine when tissue damage is already present.

Heparin mimics and fibroblast growth factor-2 fabricated nanogold composite in promoting neural differentiation of mouse embryonic stem cells

The replacement therapy or transplantation using neural cells, which differentiated from stem cells, has emerged as a promising strategy for repairing damaged neural tissues and helping functional recovery in the treatment of neural system diseases. The challenge, however, is how to control embryonic stem cell fate so that neural differentiation can be efficiently directed to enrich a neuron cell population, and meanwhile to maintain their bioactivities. This is a key question and has a very significant impact in regenerative medicine. Here we proposed a new neural-differentiation inductive nanocomposite, containing gold nanoparticles (AuNPs), poly(2-methacrylamido glucopyranose-co-3-sulfopropyl acrylate) (PMS), and basic fibroblast growth factor (FGF2), for the high efficient directional neural-specific differentiation of mouse embryonic stem cells (mESCs). In this AuNP-PMS/FGF2 composite, PMS, playing as the high-active mimic of heparin/heparan sulfate (HS), is covalently anchored to AuNPs and bound with FGF2 on the surface of nanoparticles, forming a HS/FGF2 complex nanomimics to facilitate its binding to FGF receptor (FGFR) and promote high neural-inductive activity of mESCs. The stability, bioactivity and biocompatibility of the composite are investigated in this study. The results showed that the AuNP-PMS/FGF2 composite could maintain a long-term stability at room temperature for at least 8 days, and greatly promote the neural differentiation of mESCs. Compared with the other materials, the AuNP-PMS/FGF2 composite could significantly stimulate the expression of the specific neural differentiation markers (nestin and β3-tubulin), while obviously down-regulate the mRNA production of pluripotency marker Oct-4 in mESCs. Moreover, the promotion effect of the composite on neuronal maturation marker β3-tubulin expression achieved maximally at the low concentration of FGF2 (4 ng/mL), which suggested the high efficiency of AuNP-PMS/FGF2 composite in neural differentiation of mESCs. Meanwhile, both mESCs and L929 cells showed desirable growth during the incubation with AuNP-PMS/FGF2 composite. The AuNP-PMS/FGF2 system presents a new way to achieve HS/FGF2 complex nanomimics efficiently for the neural differentiation of mESCs.

Rescue of hematopoietic stem/progenitor cells formation in plcg1 zebrafish mutant

Hematopoietic stem/progenitor cells (HSPC) in zebrafish emerge from the aortic hemogenic endothelium (HE) and migrate towards the caudal hematopoietic tissue (CHT), where they expand and differentiate during definitive hematopoiesis. Phospholipase C gamma 1 (Plcγ1) has been implicated for hematopoiesis in vivo and in vitro and is also required to drive arterial and HSPC formation. Genetic mutation in plcg1−/− (y10 allele) completely disrupts the aortic blood flow, specification of arterial fate, and HSPC formation in zebrafish embryos. We previously demonstrated that ginger treatment promoted definitive hematopoiesis via Bmp signaling. In this paper, we focus on HSPC development in plcg1−/− mutants and show that ginger/10-gingerol (10-G) can rescue the expression of arterial and HSPC markers in the HE and CHT in plcg1−/− mutant embryos. We demonstrate that ginger can induce scl/runx1 expression, and that rescued HE fate is dependent on Bmp and Notch. Bmp and Notch are known to regulate nitric oxide (NO) production and NO can induce hematopoietic stem cell fate. We show that ginger produces a robust up-regulation of NO. Taken together, we suggest in this paper that Bmp, Notch and NO are potential players that mediate the effect of ginger/10-G for rescuing the genetic defects in blood vessel specification and HSPC formation in plcg1−/− mutants. Understanding the molecular mechanisms of HSPC development in vivo is critical for understanding HSPC expansion, which will have a positive impact in regenerative medicine.

Effect of age on biomaterial-mediated in situ bone tissue regeneration

Emerging studies show the potential application of synthetic biomaterials that are intrinsically osteoconductive and osteoinductive as bone grafts to treat critical bone defects. Here, the biomaterial not only assists recruitment of endogenous cells, but also supports cellular activities relevant to bone tissue formation and function. While such biomaterial-mediated in situ tissue engineering is highly attractive, success of such an approach relies largely on the regenerative potential of the recruited cells, which is anticipated to vary with age. In this study, we investigated the effect of the age of the host on mineralized biomaterial-mediated bone tissue repair using critical-sized cranial defects as a model system. Mice of varying ages, 1-month-old (juvenile), 2-month-old (young-adult), 6-month-old (middle-aged), and 14-month-old (elderly), were used as recipients. Our results show that the bio-mineralized scaffolds support bone tissue formation by recruiting endogenous cells for all groups albeit with differences in an age-related manner. Analyses of bone tissue formation after 2 and 8 weeks post-treatment show low mineral deposition and reduced number of osteocalcin and tartrate-resistant acid phosphatase (TRAP)-expressing cells in elderly mice. Statement of SignificanceTissue engineering strategies that promote tissue repair through recruitment of endogenous cells will have a significant impact in regenerative medicine. Previous studies from our group have shown that biomineralized materials containing calcium phosphate minerals can contribute to neo-bone tissue through recruitment and activation of endogenous cells. In this study, we investigated the effect of age of the recipient on biomaterial-mediated bone tissue repair. Our results show that the age of the recipient mouse had a significant impact on the quality and quantity of the engineered neo-bone tissues, in which delayed/compromised bone tissue formation was observed in older mice. These findings are in agreement with the clinical observations that age of patients is a key factor in bone repair.

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Naïve-like State with Improved Multilineage Differentiation Potency.

Naïve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a naïve-like pluripotency with improved functionality. This chemical naïve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3β, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently naïve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. naïve-like states are outlined.

3D bioprinting – Flow cytometry as analytical strategy for 3D cell structures

The importance of 3D printing technologies increased significantly over the recent years. They are considered to have a huge impact in regenerative medicine and tissue engineering, since 3D bioprinting enables the production of cell-laden 3D scaffolds. Transition from academic research to pharmaceutical industry or clinical applications, however, is highly dependent on developing a robust and well-known process, while maintaining critical cell characteristics. Hence, a directed and systematic approach to 3D bioprinting process development is required, which also allows for the monitoring of these cell characteristics. This work presents the development of a flow cytometry-based analytical strategy as a tool for 3D bioprinting research.The development was based on a model process using a commercially available alginate-based bioink, the β-cell line INS-1E, and direct dispensing as 3D bioprinting method. We demonstrated that this set-up enabled viability and proliferation analysis. Additionally, use of an automated sampler facilitated high-throughput screenings. Finally, we showed that each process step, e.g. suspension of cells in bioink or 3D printing, cross-linking of the alginate scaffold after printing, has a crucial impact on INS-1E viability. This reflects the importance of process optimization in 3D bioprinting and the usefulness of the flow cytometry-based analytical strategy described here. The presented strategy has a great potential as a cell characterisation tool for 3D bioprinting and may contribute to a more directed process development.

Tissue engineering by decellularization and 3D bioprinting

Discarded human donor organs have been shown to provide decellularized extracellular matrix (dECM) scaffolds suitable for organ engineering. The quest for appropriate cell sources to satisfy the need of multiple cells types in order to fully repopulate human organ-derived dECM scaffolds has opened new venues for the use of human pluripotent stem cells (hPSCs) for recellularization. In addition, three-dimensional (3D) bioprinting techniques are advancing towards the fabrication of biomimetic cell-laden biomaterial constructs. Here, we review recent progress in decellularization/recellularization and 3D bioprinting technologies, aiming to fabricate autologous tissue grafts and organs with an impact in regenerative medicine.

Enhanced ex vivo expansion of adult mesenchymal stem cells by fetal mesenchymal stem cell ECM

Large-scale expansion of highly functional adult human mesenchymal stem cells (aMSCs) remains technologically challenging as aMSCs lose self renewal capacity and multipotency during traditional long-term culture and their quality/quantity declines with donor age and disease. Identification of culture conditions enabling prolonged expansion and rejuvenation would have dramatic impact in regenerative medicine. aMSC-derived decellularized extracellular matrix (ECM) has been shown to provide such microenvironment which promotes MSC self renewal and “stemness”. Since previous studies have demonstrated superior proliferation and osteogenic potential of human fetal MSCs (fMSCs), we hypothesize that their ECM may promote expansion of clinically relevant aMSCs. We demonstrated that aMSCs were more proliferative (∼1.6×) on fMSC-derived ECM than aMSC-derived ECMs and traditional tissue culture wares (TCPS). These aMSCs were smaller and more uniform in size (median ± interquartile range: 15.5 ± 4.1 μm versus 17.2 ± 5.0 μm and 15.5 ± 4.1 μm for aMSC ECM and TCPS respectively), exhibited the necessary biomarker signatures, and stained positive for osteogenic, adipogenic and chondrogenic expressions; indications that they maintained multipotency during culture. Furthermore, fMSC ECM improved the proliferation (∼2.2×), size (19.6 ± 11.9 μm vs 30.2 ± 14.5 μm) and differentiation potential in late-passaged aMSCs compared to TCPS. In conclusion, we have established fMSC ECM as a promising cell culture platform for ex vivo expansion of aMSCs.

Concise Review: Ex Vivo Expansion of Cord Blood-Derived Hematopoietic Stem and Progenitor Cells: Basic Principles, Experimental Approaches, and Impact in Regenerative Medicine

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) play key roles in the production of mature blood cells and in the biology and clinical outcomes of hematopoietic transplants. The numbers of these cells, however, are extremely low, particularly in umbilical cord blood (UCB); thus, ex vivo expansion of human UCB-derived HSCs and HPCs has become a priority in the biomedical field. Expansion of progenitor cells can be achieved by culturing such cells in the presence of different combinations of recombinant stimulatory cytokines; in contrast, expansion of actual HSCs has proved to be more difficult because, in addition to needing recombinant cytokines, HSCs seem to deeply depend on the presence of stromal cells and/or elements that promote the activation of particular self-renewal signaling pathways. Hence, there is still controversy regarding the optimal culture conditions that should be used to achieve this. To date, UCB transplants using ex vivo-expanded cells have already been performed for the treatment of different hematological disorders, and although results are still far from being optimal, the advances are encouraging. Recent studies suggest that HSCs may also give rise to nonhematopoietic cells, such as neural, cardiac, mesenchymal, and muscle cells. Such plasticity and the possibility of producing nonhematopoietic cells at the clinical scale could bring new alternatives for the treatment of neural, metabolic, orthopedic, cardiac, and neoplastic disorders. Once standardized, ex vivo expansion of human HSCs/HPCs will surely have a positive impact in regenerative medicine.

Reprogramming cells and tissue patterning via bioelectrical pathways: molecular mechanisms and biomedical opportunities.

Transformative impact in regenerative medicine requires more than the reprogramming of individual cells: advances in repair strategies for birth defects or injuries, tumor normalization, and the construction of bioengineered organs and tissues all require the ability to control large-scale anatomical shape. Much recent work has focused on the transcriptional and biochemical regulation of cell behavior and morphogenesis. However, exciting new data reveal that bioelectrical properties of cells and their microenvironment exert a profound influence on cell differentiation, proliferation, and migration. Ion channels and pumps expressed in all cells, not just excitable nerve and muscle, establish resting potentials that vary across tissues and change with significant developmental events. Most importantly, the spatiotemporal gradients of these endogenous transmembrane voltage potentials (Vmem ) serve as instructive patterning cues for large-scale anatomy, providing organ identity, positional information, and prepattern template cues for morphogenesis. New genetic and pharmacological techniques for molecular modulation of bioelectric gradients in vivo have revealed the ability to initiate complex organogenesis, change tissue identity, and trigger regeneration of whole vertebrate appendages. A large segment of the spatial information processing that orchestrates individual cells' programs toward the anatomical needs of the host organism is electrical; this blurs the line between memory and decision-making in neural networks and morphogenesis in nonneural tissues. Advances in cracking this bioelectric code will enable the rational reprogramming of shape in whole tissues and organs, revolutionizing regenerative medicine, developmental biology, and synthetic bioengineering.

Nonviral Methods for Inducing Pluripotency to Cells

The concept of inducing pluripotency to adult somatic cells by introducing reprogramming factors to them is one that has recently emerged, gained widespread acclaim and garnered much attention among the scientific community. The idea that cells can be reprogrammed, and are not unidirectionally defined opens many avenues for study. With their clear potential for use in the clinic, these reprogrammed cells stand to have a huge impact in regenerative medicine. This realization did not occur overnight but is, however, the product of many decades worth of advancements in researching this area. It was a combination of such research that led to the development of induced pluripotent stem cells as we know it today. This review delivers a brief insight in to the roots of iPS research and focuses on succinctly describing current nonviral methods of inducing pluripotency using plasmid vectors, small molecules and chemicals, and RNAs.

Large-scale production of murine embryonic stem cell-derived osteoblasts and chondrocytes on microcarriers in serum-free media

The generation of tissue-engineered constructs from stem cells for the treatment of musculoskeletal diseases may have immense impact in regenerative medicine, but there are difficulties associated with stem cell culture and differentiation, including the use of serum. Here we present serum-free protocols for the successful production of murine embryonic stem cell (mESC) derived osteoblasts and chondrocytes on CultiSpher S macroporous microcarriers in stirred suspension bioreactors. Various inoculum forms and agitation rates were investigated. Produced osteogenic cells were implanted ectopically into SCID mice and orthotopically into a murine burr-hole fracture model. Osterix, osteocalcin and collagen type I were upregulated in osteogenic cultures, while aggrecan and collagen type II were upregulated in chondrogenic cultures. Histological analysis using alizarin red S, von Kossa and alcian blue staining confirmed the presence of osteoblasts and chondrocytes, respectively in cultured microcarriers and excised tissue. Finally, implantation of derived cells into a mouse fracture model revealed cellular integration without any tumor formation. Overall, microcarriers may provide a supportive scaffold for ESC expansion and differentiation in a serum-free bioprocess for in vivo implantation. These findings lay the groundwork for the development of clinical therapies for musculoskeletal injuries and diseases using hESCs and iPS cells.

The effect of matrix characteristics on fibroblast proliferation in 3D gels

Engineering synthetic hydrogels on a molecular basis to introduce natural features that are important in instructing cell behavior is becoming increasingly crucial in biomaterial-based approaches for regenerative medicine and in cell biology to study cell–matrix interactions in three-dimensions (3D). Here, we used collagen gels and exploited the design flexibility of the biological, biochemical and physical characteristics offered by a PEG-based hydrogel system to systematically study the effect of specific extracellular microenvironments on the behavior of primary human fibroblasts in 3D. We firstly found that the proliferation profiles of fibroblasts from different patients cultured within collagen gels (3D) differed significantly from their behavior observed on tissue culture plastic (2D). Furthermore, using the biomimetic PEG-based matrix we showed that cell proliferation in 3D could be selectively manipulated via alteration of the gel characteristics. In particular, this study revealed that, in spite of matrix sensitivity to proteases (e.g. MMP) and the presence of cell-integrin binding sites, at high stiffness (elastic modulus, G′ >1200 Pa) the matrix acts as a barrier for cells cultured in 3D. Finally, a comparison between the biomimetic PEG-based and collagen gels indicated that differences in their viscoelastic behaviours, determined by the nature of network structures and cross-links, may influence the mechanism(s) cells employ to remodel their 3D extracellular microenvironment. In conclusion, these studies highlight that for proliferation in 3D, compared to 2D, cells require strategies to overcome the physical impediment posed by the matrix. We also demonstrate that by exploiting the design flexibility of the characteristics offered by these biomimetic hydrogels, it is possible to separately investigate complex aspects characterizing the cell–matrix interactions in 3D; this has the potential to have great impact in regenerative medicine, as well as in cell biology and cancer research.

Emerging peptide nanomedicine to regenerate tissues and organs

Peptide nanostructures containing bioactive signals offer exciting novel therapies of broad potential impact in regenerative medicine. These nanostructures can be designed through self-assembly strategies and supramolecular chemistry, and have the potential to combine bioactivity for multiple targets with biocompatibility. It is also possible to multiplex their functions by using them to deliver proteins, nucleic acids, drugs and cells. In this review, we illustrate progress made in this new field by our group and others using peptide-based nanotechnology. Specifically, we highlight the use of self-assembling peptide amphiphiles towards applications in the regeneration of the central nervous system, vasculature and hard tissue along with the transplant of islets and the controlled release of nitric oxide to prevent neointimal hyperplasia. Also, we discuss other self-assembling oligopeptide technology and the progress made with these materials towards the development of potential therapies.