Great Promise For Cell-based Therapies Research Articles

Safety and efficacy of umbilical cord tissue-derived mesenchymal stem cells in the treatment of patients with aging frailty: a phase I/II randomized, double-blind, placebo-controlled study

BackgroundMesenchymal stem cells (MSCs) hold a great promise for cell-based therapy in the field of regenerative medicine. In this study, we aimed to evaluate the safety and efficacy of intravenous infusion of human umbilical cord-derived MSCs (HUC-MSCs) in patients with aging frailty.MethodsIn this randomized, double-blind, placebo-controlled trial, participants diagnosed with aging frailty were randomly assigned to receive intravenous administrations of HUC-MSCs or placebo. All of serious adverse events and AEs were monitored to evaluate the safety of treatment during the 6-month follow-up. The primary efficacy endpoint was alteration of physical component scores (PCS) of SF-36 qualities of life at 6 months. The secondary outcomes including physical performance tests and pro-inflammatory cytokines, were also observed and compared at each follow-up visits. All evaluations were performed at 1 week, 1, 2, 3 and 6 months following the first intravenous infusion of HUC-MSCs.ResultsIn the MSCs group, significant improvements in PCS of SF-36 were observed from first post-treatment visit and sustained throughout the follow-up period, with greater changes compared to the placebo group (p = 0.042). EQ-VAS scores of MSCs group improved significantly at 2 month (p = 0.023) and continued until the end of the 6-month visit (p = 0.002) in comparison to the placebo group. The timed up and go (TUG) physical performance test revealed significant group difference and showed continual enhancements over 6 months (p < 0.05). MSC transplantation improved the function of 4-m walking test (4MWT) compared with the placebo group with a decrease of 2.05 s at 6 months of follow-up (p = 0.21). The measurement of grip strength revealed group difference with MSCs group demonstrating better performance, particularly at 6 months (p = 0.002). Inflammatory cytokines (TNF-α, IL-17) exhibited declines in MSCs group at 6 months compared to the placebo group (p = 0.034 and 0.033, respectively). There was no difference of incidence of AEs between the two groups.ConclusionIntravenous transplantation of HUC-MSCs is a safe and effective therapeutic approach on aging frailty. The positive outcomes observed in improving quality of life, physical performance, and reducing chronic inflammation, suggest that HUC-MSC therapy may be a promising potential treatment option for aging frailty.Trial Registration: Clinicaltrial.gov; NCT04314011; https://clinicaltrials.gov/ct2/show/NCT04314011.

An Optogenetic-Controlled Cell Reprogramming System for Driving Cell Fate and Light-Responsive Chimeric Mice.

Pluripotent stem cells (PSCs) hold great promise for cell-based therapies, disease modeling, and drug discovery. Classic somatic cell reprogramming to generate induced pluripotent stem cells (iPSCs) is often achieved based on overexpression of transcription factors (TFs). However, this process is limited by side effect of overexpressed TFs and unpredicted targeting of TFs. Pinpoint control over endogenous TFs expression can provide the ability to reprogram cell fate and tissue function. Here, a light-inducible cell reprogramming (LIRE) system is developed based on a photoreceptor protein cryptochrome system and clustered regularly interspaced short palindromic repeats/nuclease-deficient CRISPR-associated protein 9for induced PSCs reprogramming. This system enables remote, non-invasive optogenetical regulation of endogenous Sox2and Oct4loci to reprogram mouse embryonic fibroblasts into iPSCs (iPSCLIRE ) under light-emitting diode-based illumination. iPSCLIRE cells can be efficiently differentiated into different cells by upregulating a corresponding TF. iPSCLIRE cells are used for blastocyst injection and optogenetic chimeric mice are successfully generated, which enables non-invasive control of user-defined endogenous genes in vivo, providing a valuable tool for facile and traceless controlled gene expression studies and genetic screens in mice. This LIRE system offers a remote, traceless, and non-invasive approach for cellular reprogramming and modeling of complex human diseases in basic biological research and regenerative medicine applications.

Surface coating materials regulates the attachment and differentiation of mouse embryonic stem cell derived embryoid bodies into mesoderm at culture conditions.

Pluripotent stem cells as a promising cell source with unlimited proliferation and differentiation capacity hold great promise for cell-based therapies in regenerative medicine. Establishment of appropriate culture conditions might enable the control of cellular fate decision in cell culture. Transfer of three-dimensional (3D) embryoid bodies to two-dimensional (2D) monolayer culture systems for initiation of cell differentiation and specialization requires an adaptation of cells which can be managed by extracellular matrix (ECM) materials. Here we compare the characteristics of four different cell culture coating materials and their effect on attachment and differentiation of cells spreading from mouse embryonic stem cell (mESC) derived embryoid bodies (EBs) in mesoderm inducing culture conditions. Atomic force microscope (AFM) and scanning electron microscope (SEM) analysis along with Water Contact Angle technique were used to analyze physical properties of ECM materials and to evaluate cellular behavior on surfaces. Cell migration and differentiation were performed initially by using mesoderm inducing culture conditions and then three germ layer specification conditions. We investigated properties of coating materials such as roughness and wettability control cell attachment, migration and differentiation of mESCs. Matrigel-Gelatin combination is suitable for cell attachment and migration of cells spreading from 3D EBs followed by transfer onto coated surfaces. Matrigel-Gelatin coating enhanced differentiation of cells into mesoderm like cells via EMT process. Our data demonstrated that the Matrigel-Gelatin combination as a cell culture coating matrix might serve as a suitable platform to transfer EBs for differentiation and might influence pluripotent stem cell fate decision into mesoderm and further mesoderm derivative cell populations. The online version contains supplementary material available at 10.1007/s10616-022-00529-z.

Induced Pluripotent Stem Cells as a Tool for Modeling Hematologic Disorders and as a Potential Source for Cell-Based Therapies.

The breakthrough in human induced pluripotent stem cells (hiPSCs) has revolutionized the field of biomedical and pharmaceutical research and opened up vast opportunities for drug discovery and regenerative medicine, especially when combined with gene-editing technology. Numerous healthy and patient-derived hiPSCs for human disease modeling have been established, enabling mechanistic studies of pathogenesis, platforms for preclinical drug screening, and the development of novel therapeutic targets/approaches. Additionally, hiPSCs hold great promise for cell-based therapy, serving as an attractive cell source for generating stem/progenitor cells or functional differentiated cells for degenerative diseases, due to their unlimited proliferative capacity, pluripotency, and ethical acceptability. In this review, we provide an overview of hiPSCs and their utility in the study of hematologic disorders through hematopoietic differentiation. We highlight recent hereditary and acquired genetic hematologic disease modeling with patient-specific iPSCs, and discuss their applications as instrumental drug screening tools. The clinical applications of hiPSCs in cell-based therapy, including the next-generation cancer immunotherapy, are provided. Lastly, we discuss the current challenges that need to be addressed to fulfill the validity of hiPSC-based disease modeling and future perspectives of hiPSCs in the field of hematology.

Chemically Defined, Clinical-Grade Cryopreservation of Human Adipose Stem Cells.

Adipose-derived stem cells (ASCs) reside in the stromal compartment of adipose tissue and can be easily harvested in large quantities through a clinically safe liposuction procedure. ASCs do not induce immunogenic reactions and rather exert immunosuppressive effects. Therefore, they can be used for both autologous and allogeneic transplantations. They hold great promise for cell-based therapies and tissue engineering. A prerequisite to the realization of this promise is the development of successful cryopreservation methods for ASCs. In this chapter, we describe a xeno-free- and chemically defined cryopreservation protocol, which can be used for various clinical applications of ASCs.

Induction of Hepatic Regeneration in an Experimental Model Using Hepatocyte-Differentiated Mesenchymal Stem Cells

Mesenchymal stem cell (MSC)-based liver tissue engineering on nanofibrous scaffold holds great promise for cell-based therapy in liver injuries and end-stage liver failure treatments. MSCs were generated from umbilical cord blood. Hepatogenic differentiation was induced on two-dimensional (2D) and three-dimensional (3D) culture system and characterized by morphology, scanning electron microscopy, immunocytochemistry, and gene expression. Albumin and α-1 antitrypsin (AAT) in culture supernatants were measured. Differentiated cells were administered intravenous into a murine model of carbon tetra induced liver cirrhosis. After 12 weeks of injection, liver pathology was examined. The hepatogenic differentiated MSCs stained positively for albumin, alpha fetoprotein, HepPar1, cytokeratin 7 and 18, and OV6 with more mature cells, hexagonal in shape with central nuclei forming large sheets in groups in 3D culture system. AAT secretion and indocyanine green uptake were significantly increased in 3D system. In experimental model, MSC-3D treated group exhibited maximal restoration of liver architecture with absent septal fibrosis and marked improvement of alanine transaminase (ALT) and aspartate transaminase (AST), and mild increase in albumin. Both 3D and 2D culture system are effective in functional hepatogenic differentiation from MSCs and serve as a vehicle in liver tissue engineering. In vivo hepatogenic differentiation is more effective on 3D scaffold, with better functional recovery.

Genome-scale screens identify JNK-JUN signaling as a barrier for pluripotency exit and endoderm differentiation.

Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five JNK/JUN family genes as key barriers of DE differentiation. The JNK/JUN pathway does not act through directly inhibiting the DE enhancers. Instead JUN co-occupies ESC enhancers with OCT4, NANOG and SMAD2/3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2/3 chromatin binding from ESC to DE enhancers. Therefore, the JNK/JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine.

Differential Expression of PPARγ and CHOP‐10 During Adipogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) hold great promise for cell‐based therapy and regenerative medicine owing to their multilineage differentiation potential that ranges from osteogenic to cardiac lineages. Osteogenic and adipogenic differentiation of MSCs in particular is of great interest since any imbalance that perturbs the regulation of osteogenesis and adipogenesis is typically associated with diseases such as obesity, osteoporosis, and other age‐related diseases. In our project, we sought to profile the gene expression pattern of key osteogenic and adipogenic transcription factors during differentiation using time course experiments. Adipogenesis was induced in passage 4 human bone marrow derived mesenchymal stem cells (BM‐MSCs) seeded with a density of 6579 cells/cm2 and RNA was collected at Day 0 (control), Day 3, Day 7, and Day 14 after differentiation to analyze the expression level of master adipogenic transcription factor peroxisome proliferator activated receptor‐γ (PPARγ) and negative adipogenic factor CHOP‐10. Our preliminary data suggests that compared to control (Day 0, undifferentiated) samples, PPARγ expression increases about 20‐fold, 22‐fold, and 168‐fold at Day 3, 7, 14 respectively. In contrast, expression of CHOP‐10, which is a negative regulator of adipogenesis, is upregulated approximately 2.4‐fold at Day 3, then downregulated 1.13‐fold at Day 7, and again increased 1.1‐fold at Day 14 respectively. Future work will compare these results and expression pattern of other adipogenic transcription factors to MSC differentiation in a 3D culture system.Support or Funding InformationFunding for these projects are supported by Wright State University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Immunohematology Mesenchymal Stromal Cell-based Therapy: From Research to Clinic.

Mesenchymal stromal cells (MSC) are nonhematopoietic cells that can be isolated from several adult and fetal tissues. MSC present specific features as the capacity to support hematopoiesis and to regulate immune response. Thus, the use of MSC as a cell therapeutic product in the field of immune-hematology is of great importance. In this review, we focused on human MSC and discussed their immune-hematologic properties and their translation toward therapeutic clinical applications. Thus, these features hold great promise for cell-based therapy and are of important relevance for the field.

Interactions of human embryonic stem cell‐derived cardiovascular progenitor cells with immobilized extracellular matrix proteins

Human embryonic stem cell-derived cardiovascular progenitor cells (hESC-CVPCs) hold great promise for cell-based therapies of heart diseases. However, little is known about their niche microenvironment and in particular the required extracellular matrix (ECM) components. Here we screened combinations of surface-immobilized ECM proteins to identify substrates that support the attachment and survival of hESC-CVPCs. Covalent immobilization of ECM proteins laminin (Lm), fibronectin (Fn), collagen I (CI), collagen III (CIII), and collagen IV (CIV) in multiple combinations and concentrations was achieved by reductive amination on transparent acetaldehyde plasma polymer (AAPP) interlayer coatings. We identified that CI, CIII, CIV, and Fn and their combinations were important for hESC-CVPC attachment and survival, while Lm was dispensable. Moreover, for coatings displaying single ECM proteins, CI and CIII performed better than CIV and Fn, while coatings displaying the combined ECM proteins CIII + CIV and Fn + CIII + CIV at 100 µg/mL were comparable to Matrigel in regard to supporting hESC-CVPC attachment and viability. Our results identify ECM proteins required for hESC-CVPCs and demonstrate that coatings displaying multiple immobilized ECM proteins offer a suitable microenvironment for the attachment and survival of hESC-CVPCs. This knowledge contributes to the development of approaches for maintaining hESC-CVPCs and therefore to advances in cardiovascular regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1094-1104, 2017.

The Development of Macrophage-Mediated Cell Therapy to Improve Skeletal Muscle Function after Injury.

Skeletal muscle regeneration following acute injury is a multi-step process involving complex changes in tissue microenvironment. Macrophages (MPs) are one of the key cell types involved in orchestration and modulation of the repair process. Multiple studies highlight the essential role of MPs in the control of the myogenic program and inflammatory response during skeletal muscle regeneration. A variety of MP phenotypes have been identified and characterized in vitro as well as in vivo. As such, MPs hold great promise for cell-based therapies in the field of regenerative medicine. In this study we used bone-marrow derived in vitro LPS/IFN-y-induced M1 MPs to enhance functional muscle recovery after tourniquet-induced ischemia/reperfusion injury (TK-I/R). We detected a 15% improvement in specific tension and force normalized to mass after M1 (LPS/IFN-γ) MP transplantation 24 hours post-reperfusion. Interestingly, we found that M0 bone marrow-derived unpolarized MPs significantly impaired muscle function highlighting the complexity of temporally coordinated skeletal muscle regenerative program. Furthermore, we show that delivery of M1 (LPS/IFN-γ) MPs early in regeneration accelerates myofiber repair, decreases fibrotic tissue deposition and increases whole muscle IGF-I expression.

193. Establishment of an Optimization Procedure for Large-Scale Production of Patient-Specific Cardiomyocytes from hiPSCs

Cardiovascular diseases are a leading cause of morbidity and mortality worldwide. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), with their ability of indefinite self-renewal and capability to differentiate into all three germ layers, provide a powerful tool in developmental biology, for drug screening, disease modeling, and potentially cell replacement therapy for diseases, such as myocardial infarction and heart failure. Unlike hESCs, hiPSCs are generated from reprogrammed somatic cells without using of human embryos and thus hold a great promise for cell-based therapy as they avoid ethical and immune rejection issues. However, due to scalability limitation of current hiPSC adherent culture platform and potential variation of culture and differentiation conditions among hiPSC lines, production of hiPSC-derived cells to a scale sufficient for therapeutic application in a timely manner has been challenging. Previously we have developed a scalable, defined, and cGMP-compliant suspension culture system for hPSC expansion and cardiac differentiation using spinner flasks by optimizing critical culture parameters we identified. In the present study, we tested the feasibility of applying our suspension culture platform and optimization strategy to establish a process for scale-up production of cardiomyocytes (CM) from individual hiPSC lines in a timely manner. We optimized cell seeding density, agitation rate, concentration of differentiation inducer, and timing for differentiation induction to determine an optimal condition for large-scale CM production. The process from suspension adaptation, differentiation optimization to CM production was accomplished in 3 months. With optimized culture and differentiation conditions, we showed that the hiPSC suspension culture can be serially passaged and expanded, and maintained normal karyotype. At a scale of 500mL suspension culture, 4-5×108 cells with 70-90% CM purity were manufactured. In summary, we demonstrated the feasibility of applying the suspension culture platform and optimization strategy we developed to establish a production process for individual hiPSC line to manufacture CM in a large scale in a timely manner.

Differentiation of Human Adipose-Derived Stem Cells into Neuron-Like Cells Which Are Compatible with Photocurable Three-Dimensional Scaffolds

Multipotent human adipose-derived stromal/stem cells (hADSCs) hold a great promise for cell-based therapy for many devastating human diseases, such as spinal cord injury and stroke. If exogenous hADSCs can be cultured in a three-dimensional (3D) scaffold with effective proliferation and differentiation capacity, it will better mimic the in vivo environment, which will have profound impact on the therapeutic application of hADSCs. In this study, a group of elastic-dominant, porous bioscaffolds from photocurable chitosan and gelatin were fabricated and proven to be biocompatible with both hADSCs and hADSC-derived neuron-like cells (hADSC-NLCs) in vitro. The identity of harvested hADSCs was confirmed by their positive immunostaining of mesenchymal stem cell surface markers, CD29, CD44, and CD105, and also positive expression of stem markers, Sox-2, Oct-4, c-Myc, Nanog, and Klf4. Their multipotency was further confirmed by trilineage differentiation of hADSCs toward adipocyte, osteoblast, and chondrocyte. It was found that hADSCs could be conditioned to differentiate into neurons in vitro as determined by immunostaining the markers of Tuj1, MAP2, NeuN, and Synapsin. The hADSCs and hADSC-NLCs were proven to be biocompatible with 3D scaffold, which actually facilitated the proliferation and differentiation of hADSCs in vitro, by MTT assay and their neuronal gene expression profiling. Moreover, hADSC-NLCs, which were mixed with 3D scaffold and transplanted into traumatic brain injury mouse model, survived in vivo and led to the better repair of the damaged brain area. The immunohistochemical studies revealed that 3D scaffold indeed improved the viability of transplanted cells, their ability to incorporate into the in vivo neural circuit, and their capacity for tissue repair. This study indicates that hADSCs would have great therapeutic application potential as seeding cells for in vivo transplantation to treat various neurological diseases when co-applied with porous chitosan/gelatin bioscaffolds.

Trans-differentiation of human mesenchymal stem cells generates functional hepatospheres on poly(l-lactic acid)-co-poly(ε-caprolactone)/collagen nanofibrous scaffolds.

Mesenchymal stem cell (MSC)-based liver tissue engineering on nanofibrous scaffold holds great promise for cell-based therapy in liver injuries and end-stage liver failure treatments. We investigated the hepatic trans-differentiation potential of human MSCs on a biocomposite poly(l-lactic acid)-co-poly (ε-caprolactone)/collagen (PLACL/collagen) nanofibrous scaffold. The nanofibrous scaffolds comprised of PLACL, collagen and a PLACL/collagen blend (2 : 1) were fabricated by electrospinning and also evaluated for fiber morphology, surface wettability, functional groups, porosity and tensile properties. Hepatic trans-differentiation of human bone marrow-derived MSCs (hMSCs) was carried out on these scaffolds over a period of 28 days using sequential induction with hepatogenic growth factors. Hepatogenesis was confirmed by scanning electron microscopy (SEM), cell phenotype tracking dye expression, quantitative expression of hepatic genes, immunofluorescence staining of hepatocyte-specific markers and albumin release. The results proved that the porous PLACL/collagen nanofibrous scaffold supported enhanced hMSC proliferation and hepatic trans-differentiation compared to individual PLACL and collagen scaffolds as well as a monolayer culture on tissue culture plate (TCP). Interestingly, hMSC-derived hepatocyte-like cells on PLACL/collagen nanofibrous scaffolds could aggregate to form functional 'hepatospheres' similar to normal hepatic spheroids. The present study concludes that PLACL/collagen nanofibrous scaffolds are potentially biomimetic and upon sequential induction with hepatogenic growth factors/cytokines, it augments trans-differentiation of hMSCs towards functional hepatosphere formation. Such bioengineered nanofibrous scaffold hepatic construct provides a promising approach for cellular therapy of damaged livers in end-stage liver failure treatments.

Mesenchymal Stem or Stromal Cells from Amnion and Umbilical Cord Tissue and Their Potential for Clinical Applications

Mesenchymal stem or stromal cells (MSC) have proven to offer great promise for cell-based therapies and tissue engineering applications, as these cells are capable of extensive self-renewal and display a multilineage differentiation potential. Furthermore, MSC were shown to exhibit immunomodulatory properties and display supportive functions through parakrine effects. Besides bone marrow (BM), still today the most common source of MSC, these cells were found to be present in a variety of postnatal and extraembryonic tissues and organs as well as in a large variety of fetal tissues. Over the last decade, the human umbilical cord and human amnion have been found to be a rich and valuable source of MSC that is bio-equivalent to BM-MSC. Since these tissues are discarded after birth, the cells are easily accessible without ethical concerns.

Dynamic HoxB4-regulatory network during embryonic stem cell differentiation to hematopoietic cells

AbstractEfficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies to treat hematologic diseases. To date, HoxB4 remains the most effective transcription factor (TF) the overexpression of which in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop more efficient protocols for in vitro derivation of HSCs. In the present study, we performed global gene-expression profiling and ChIP coupled with deep sequencing at 4 stages of the HoxB4-mediated ESC differentiation toward HSCs. Joint analyses of ChIP/deep sequencing and gene-expression profiling unveiled several global features of the HoxB4 regulatory network. First, it is highly dynamic and gradually expands during the differentiation process. Second, HoxB4 functions as a master regulator of hematopoiesis by regulating multiple hematopoietic TFs and chromatin-modification enzymes. Third, HoxB4 acts in different combinations with 4 other hematopoietic TFs (Fli1, Meis1, Runx1, and Scl) to regulate distinct sets of pathways. Finally, the results of our study suggest that down-regulation of mitochondria and lysosomal genes by HoxB4 plays a role in the impaired lymphoid lineage development from ESC-derived HSCs.

Understanding the Molecular Circuitry of Cell Lineage Specification in the Early Mouse Embryo

Pluripotent stem cells hold great promise for cell-based therapies in regenerative medicine. However, critical to understanding and exploiting mechanisms of cell lineage specification, epigenetic reprogramming, and the optimal environment for maintaining and differentiating pluripotent stem cells is a fundamental knowledge of how these events occur in normal embryogenesis. The early mouse embryo has provided an excellent model to interrogate events crucial in cell lineage commitment and plasticity, as well as for embryo-derived lineage-specific stem cells and induced pluripotent stem (iPS) cells. Here we provide an overview of cell lineage specification in the early (preimplantation) mouse embryo focusing on the transcriptional circuitry and epigenetic marks necessary for successive differentiation events leading to the formation of the blastocyst.

The effect of type I collagen on osteochondrogenic differentiation in adipose-derived stromal cells in vivo

Recent studies have demonstrated that adipose-derived adult stromal cells (ADASCs) offer great promise for cell-based therapies due to their ability to differentiate towards bone, cartilage and fat [corrected] The objective of this study was to investigate whether type I collagen would elicit in vivo bone formation of passaged rat adipose-derived adult stromal cells (ADASC) placed extraskeletally. After expansion for 1-4 passages (P), cells were incubated in osteogenic medium containing dexamethasone, ascorbic acid and beta-glycerol phosphate for 2-4 weeks. Undifferentiated cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and von Kossa staining as well as by gene expression of ALP, osteopontin (OP), osteonectin (ON), osteocalcin (OC), collagen I (colI), collagen II (colII), bone sialoprotein (BSP), periostin (Postn), runx2, osterix (Osx), sox9, msx1 and msx2. Diffusion chambers were filled with 1x10(6) cells mixed with or without type I collagen gel and implanted subcutaneously into rats. Controls included chambers exposed to (1) undifferentiated cells (with or without collagen, (2) collagen without cells and (3) empty chambers (n=5 per group). Four weeks after implantation, in vivo bone and cartilage formation was demonstrated in implants containing 4-week osteo-induced P1 and P4 cells wrapped in the collagen gel, as confirmed by Goldner's trichrome and Alcian blue staining, respectively. Newly formed bone stained positive for type I collagen. Control implants had no bone or cartilage and were primarily filled with fibrous tissue at that time interval. Recent studies have demonstrated that ADASC offer great promise for cell-based therapies because of their ability to differentiate toward bone, cartilage and fat. However, the influence of different matrices on the in vivo osteogenic capability of ADASC is not fully understood. These findings suggest that type I collagen may support the survival and expression of osteogenic and chondrogenic phenotypes in passaged rat ADASC in vivo.

Efficient propagation of single cells accutase‐dissociated human embryonic stem cells

Human embryonic stem cells (hESCs) hold great promise for cell-based therapies and drug screening applications. However, growing and processing large quantities of undifferentiated hESCs is a challenging task. Conventionally, hESCs are passaged as clusters, which can limit their growth efficiency and use in downstream applications. This study demonstrates that hESCs can be passaged as single cells using Accutase, a formulated mixture of digestive enzymes. In contrast to trypsin treatment, Accutase treatment does not significantly affect the viability and proliferation rate of hESC dissociation into single cells. Accutase-dissociated single cells can be separated by FACS and proliferate as fully pluripotent hESCs. An Oct4-eGFP reporter construct engineered into hESCs was used to monitor the pluripotency of hESCs passaged with Accutase. Compared to collagenase-passaged hESCs, Accutase-treated cultures contained a larger proportion of undifferentiated (Oct4-positive) cells. Additionally, Accutase-passaged undifferentiated hESCs could be grown as monolayers without the need for monitoring and/or selection for quality hESC colonies.

Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells

Recent studies have demonstrated that adipose-derived mesenchymal cells (AMCs) offer great promise for cell-based therapies because of their ability to differentiate toward bone, cartilage, and fat. Given that cartilage is an avascular tissue and that mesenchymal cells experience hypoxia during prechondrogenic condensation in endochondral ossification, the goal of this study was to understand the influence of oxygen tension on AMC differentiation into bone and cartilage. In vitro chondrogenesis was induced using a three-dimensional micromass culture model supplemented with TGF-beta1. Collagen II production and extracellular matrix proteoglycans were assessed with immunohistochemistry and Alcian blue staining, respectively. Strikingly, micromasses differentiated in reduced oxygen tension (2% O(2)) showed markedly decreased chondrogenesis. Osteogenesis was induced using osteogenic medium supplemented with retinoic acid or vitamin D and was assessed with alkaline phosphatase activity and mineralization. AMCs differentiated in both 21 and 2% O(2) environments. However, osteogenesis was severely diminished in a low-oxygen environment. These data demonstrated that hypoxia strongly inhibits in vitro chondrogenesis and osteogenesis in AMCs.