Pre-differentiated Cells Research Articles

Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels

Physiologically relevant hepatic cell culture models must be based on three-dimensional (3D) culture of human cells. However, liver cells are generally cultured in two-dimensional (2D) format that deviates from the normal in vivo morphology. We generated 3D culture environment for HepaRG liver progenitor cells using wood-derived nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG) hydrogels. Culture of undifferentiated HepaRG cells in NFC and HG hydrogels induced formation of 3D multicellular spheroids with apicobasal polarity and functional bile canaliculi-like structures, structural hallmarks of the liver tissue. Furthermore, hepatobiliary drug transporters, MRP2 and MDR1, were localized on the canalicular membranes of the spheroids and vectorial transport of fluorescent probes towards the biliary compartment was demonstrated. Cell culture in 3D hydrogel supported the mRNA expression of hepatocyte markers (albumin and CYP3A4), and metabolic activity of CYP3A4 in the HepaRG cell cultures. On the contrary, the 3D hydrogel cultures with pre-differentiated HepaRG cells showed decreasing expression of albumin and CYP3A4 transcripts as well as CYP3A4 activity. It is concluded that NFC and HG hydrogels expedite the hepatic differentiation of HepaRG liver progenitor cells better than the standard 2D culture environment. This was shown as improved cell morphology, expression and localization of hepatic markers, metabolic activity and vectorial transport. The NFC and HG hydrogels are promising materials for hepatic cell culture and tissue engineering.

Engraftment of pre-differentiated stem cells into cardiomyocytes in an animal model of ischemic cardiopathy

Stem cell therapy for cardiac infarct regeneration has been widely used in clinical research. Despite the fact that important advances in this field have been reached, the observed recovery does not demonstrate new cardiac muscle formation. Benefits have been observed due to an improvement in neovascularisation. The main objective of this study was to determine if pre-differentiated stem cells into cells of myocardic lineage are capable of engraftment in animal models with induced cardiac infarct and are capable of truly differentiating into myocardiocytes. Bone marrow rat stem cells were pre-differentiated with 5-AZ. After 4 weeks, pre-differentiated stem cells express muscarinic 1, 2 and β adrenergic 2 receptors. Also, proteins such as sarcomeric α-actin, cardiac myosin heavy chain, desmin and vimentin were detected by immunocytochemistry. Cells were transplanted intracardialy in an ischemic cardiac rat model. Pre-differentiated or non differentiated cells were transplanted after 4 weeks post infarct induction. Histopathology of the hearts was made 2 weeks after cell transplantation. Typical granulated tissue, scare formation and neovascularisation were observed in both groups. However, in those hearts from rats inoculated with pre-differentiated cells many appeared atypical and were α-actin sarcomeric positive. These events suggest that pre-differentiated cells conserve some muscle characteristic traits in situ that at least last for two weeks after transplantation.

Generation of osteochondral tissue constructs with chondrogenically and osteogenically predifferentiated mesenchymal stem cells encapsulated in bilayered hydrogels

This study investigated the ability of chondrogenic and osteogenic predifferentiation of mesenchymal stem cells (MSCs) to play a role in the development of osteochondral tissue constructs using injectable bilayered oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel composites. We hypothesized that the combinatorial approach of encapsulating cell populations of both chondrogenic and osteogenic lineages in a spatially controlled manner within bilayered constructs would enable these cells to maintain their respective phenotypes via the exchange of biochemical factors even without the influence of external growth factors. During monolayer expansion prior to hydrogel encapsulation, it was found that 7 (CG7) and 14 (CG14) days of MSC exposure to TGF-β3 allowed for the generation of distinct cell populations with corresponding chondrogenic maturities as indicated by increasing aggrecan and type II collagen/type I collagen expression. Chondrogenic and osteogenic cells were then encapsulated within their respective (chondral/subchondral) layers in bilayered hydrogel composites to include four experimental groups. Encapsulated CG7 cells within the chondral layer exhibited enhanced chondrogenic phenotype when compared to other cell populations based on stronger type II collagen and aggrecan gene expression and higher glycosaminoglycan-to-hydroxyproline ratios. Osteogenic cells that were co-cultured with chondrogenic cells (in the chondral layer) showed higher cellularity over time, suggesting that chondrogenic cells stimulated the proliferation of osteogenic cells. Groups with osteogenic cells displayed mineralization in the subchondral layer, confirming the effect of osteogenic predifferentiation. In summary, it was found that MSCs that underwent 7days, but not 14days, of chondrogenic predifferentiation most closely resembled the phenotype of native hyaline cartilage when combined with osteogenic cells in a bilayered OPF hydrogel composite, indicating that the duration of chondrogenic preconditioning is an important factor to control. Furthermore, the respective chondrogenic and osteogenic phenotypes were maintained for 28days in vitro without the need for external growth factors, demonstrating the exciting potential of this novel strategy for the generation of osteochondral tissue constructs for cartilage engineering applications.

Elimination of both cancer stem cells and progenitor cells as essential for tumor eradication.

e22179 Background: Cancer stem cells (CSC) have been identified in several malignancies. Elimination of CSCs requires precise identification of markers and development of mechanisms to target them specifically. It is possible that in spite of elimination of CSCs, the tumor may recur. Dedifferentiation of progenitor cells may help replenish cancer stem cells. Methods: A mathematical model of a cancer stem cell system with four layers has been constructed to reflect the hierarchy of cancer stem cells, progenitor cells and, transit-amplifying cells and pre-differentiated cells. This model is constructed in the lines of the phase space model described by Kirkland. Dedifferentiation parameters were introduced to allow for dedifferentiation when the stem cell pool receded below 40% of its normal size. The effects of stem cell antagonists (SCA) and differentiating agents (DA) were simulated to study the perturbations caused by them on the growth of the tumor. Results: SCAs when used alone result in the establishment of a new equilibrium across different layers within the tumor by de-differentiation. DAs which act only on progenitor cells cause an increase in the number of cells in the subsequent layers and contribute to an increase in the size of the tumor. DAs which act only on the pre-differentiated layer of cells move cells from this layer into a terminally differentiated layer of cells which results in a new but lower equilibrium. DAs which act on the progenitor, transit amplifying and pre-differentiated cells cause the establishment of a new equilibrium which is lower than that of all agents which act on individual layers of cells. A combination of an SCA and a DA which acts on multiple layers eliminates the re-establishment of a new equilibrium and leads to decline in cell populations in all the layers, thus reducing the possibility of recurrence. Conclusions: SCAs when used individually are not effective in eliminating the tumor. DAs which act only on progenitor cells can fuel tumor growth but DAs which act on multiple layers have a profound effect in slowing the growth of the tumor but do not eliminate the tumor. This model implies that a combination of an SCA and a DA which acts on multiple layers may have a better chance at eliminating the tumor.

Neuron and beta‐cell evolution: Learning about neurons is learning about beta‐cells

Neurons and insulin-producing pancreatic beta-cells share multiple common features although they arise from different germ layers 1. In analogy to neurons, beta-cells release insulin by membrane depolarization reminiscent of synaptic neurotransmitter release in neurons and produce the neurotransmitter gamma aminobutyric acid (GABA) in synaptic-like vesicles. Although expression of many individual genes has been shown in neurons and beta-cells, an estimation of the total amount of genes commonly expressed in these cell types has just been addressed recently 2, 3. In a genome-wide mRNA expression scan, profiles of human and rodent beta-cells were compared to non-beta-cell tissues to find genes selectively expressed in beta-cells 2. Interestingly, 15% of these conserved beta-cell markers were also expressed in neuronal tissue coding for proteins involved in neurotransmitter transport, synaptic vesicle formation and brain development 2. To explain why ectodermal-derived neurons and endodermal-derived beta-cells share many common features, Arntfield and van der Kooy have recently speculated that beta-cells may have “borrowed from the brain”, i.e. their ancestors have deployed parts of the neuronal developmental program thereby creating an example of convergent evolution 1. This idea is consistent with the fact that multiple transcription factors are expressed in the neuronal and the pancreatic endocrine lineage including Neurogenin 3 (Ngn3) and NeuroD, both essential for brain and pancreas development. Arntfield et al. have further suggested that the evolution of (modern) beta-cells may have resulted from stochastic effects, i.e. mutation, activating neuronal master transcription factors in gut cells 1. However it remains unclarified which cell type may have first adopted the neuronal program, i.e. a more unspecified gut cell or a pre-differentiated endocrine cell. Recently it has been suggested that beta-cell progenitors co-opt the neural program rather late during their differentiation based on observations on the level of epigenetics 3. Interestingly, neuronal genes are still repressed in multipotent pancreatic progenitors that can give rise to exo- or endocrine tissue suggesting that an activation of neuronal gene activity occured later in the endocrine lineage 3. This would indicate that a pre-differentiated multipotent pancreatic progenitor adopted the neuronal program rather than a more unspecified cell. The notion that neurons and beta-cells are similar has stimulated scientists to transfer principles from neurobiology to understand beta-cell function and to find drugs to support beta-cell function under stress conditions as observed in type 2 diabetes mellitus. For example, screening of a chemical library for neurogenic activators has recently led to the identification of a potent inducer of NeuroD, supporting insulin production in pancreatic beta-cells 4. Moreover, known neuroprotective mechanisms may also be essential in beta-cells as observed in the case of the Parkinson-related antioxidant protein DJ-1 5. DJ-1 preserves mitochondrial integrity in dopaminergic neurons that are vulnerable to reactive oxygen species. In analogy to neurons, DJ-1 also protects the mitochondrial function of beta-cells suggesting that beta-cells and neurons share similar oxidative stress defence mechanisms. Thus, the study of common principles in neurons and beta-cells may provide a basis for prevention and treatment of diabetes mellitus and neurodegenerative diseases.

Winner for outstanding research in the Ph.D. category for the 2013 society for biomaterials meeting and exposition, April 10–13, 2013, Boston, Massachusetts

Modular tissue engineering applies biomaterials-based approaches to create discrete cell-seeded microenvironments, which can be further assembled into larger constructs for the repair of injured tissues. In the current study, we embedded human bone marrow-derived mesenchymal stem cells (MSC) and human adipose-derived stem cells (ASC) in collagen/fibrin (COL/FIB) and collagen/fibrin/hydroxyapatite (COL/FIB/HA) microbeads, and evaluated their suitability for bone tissue engineering applications. Microbeads were fabricated using a water-in-oil emulsification process, resulting in an average microbead diameter of approximately 130 ± 25 μm. Microbeads supported both cell viability and cell spreading of MSC and ASC over 7 days in culture. The embedded cells also began to remodel and compact the microbead matrix as demonstrated by confocal reflectance microscopy imaging. After two weeks of culture in media containing osteogenic supplements, both MSC and ASC deposited calcium mineral in COL/FIB microbeads, but not in COL/FIB/HA microbeads. There were no significant differences between MSC and ASC in any of the assays examined, suggesting that either cell type may be an appropriate cell source for orthopedic applications. This study has implications in the creation of defined microenvironments for bone repair, and in developing a modular approach for delivery of pre-differentiated cells.

Depolarization alters phenotype, maintains plasticity of pre-differentiated mesenchymal stem cells

Depolarization alters phenotype, maintains plasticity of pre-differentiated mesenchymal stem cells

Winner of the 2013 young investigator award for the society for biomaterials annual meeting and exposition, April 10–13, 2013, Boston, Massachusetts

AbstractDevitalization and demineralization processing of biodegradable polymer and extracellular matrix (ECM) hybrid constructs was explored for the effect on the retention of ECM components and construct osteogenicity. Hybrid constructs were generated by seeding osteogenically predifferentiated rat mesenchymal stem cells (MSCs) onto electrospun poly(ε‐caprolactone) fiber meshes and culturing in osteogenic medium for 12 or 16 days within a flow perfusion bioreactor to create an ECM coating. The resulting constructs were then either devitalized (using a freeze‐thaw or a detergent technique), devitalized and demineralized, or left untreated, and subsequently characterized for DNA, glycosaminoglycan, collagen, and calcium content. The osteogenicity of each construct was investigated by culturing MSCs on the hybrid constructs within a flow perfusion bioreactor for 4, 8, and 12 days in osteogenic medium. Histological staining demonstrated that devitalization via the freeze–thaw method retained the thickest coating of ECM components within the constructs. Demineralization and devitalization processing of ECM coated constructs resulted in a decrease in their osteogenicity. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

253 Synergistic property of triple combination pre-differentiated cell transplantation for urethral sphincter regeneration

253 Synergistic property of triple combination pre-differentiated cell transplantation for urethral sphincter regeneration

Tenogenic differentiation of equine adipose-tissue-derived stem cells under the influence of tensile strain, growth differentiation factors and various oxygen tensions

Mesenchymal stem cells have become extremely interesting for regenerative medicine and tissue engineering in the horse. Stem cell therapy has been proven to be a powerful and successful instrument, in particular for the healing of tendon lesions. We pre-differentiated equine adipose-tissue-derived stem cells (ASCs) in a collagen I gel scaffold by applying tensile strain, growth differentiation factors (GDFs) and various oxygen tensions in order to determine the optimal conditions for in vitro differentiation toward the tenogenic lineage. We compared the influence of 3% versus 21% oxygen tension, the use of GDF 5, GDF 6 and GDF 7 and the application of uniaxial tensile strain versus no mechanical stimulation on differentiation results as evaluated by cell morphology and by the expression of the tendon-relevant genes collagen I, collagen III, cartilage oligomeric matrix protein and scleraxis. The best results were obtained with an oxygen tension of 21%, tensile stimulation and supplementation with GDF 5 or GDF 7. This approach raises the hope that the in vivo application of pre-differentiated stem cells will improve healing and recovery time in comparison with treatment involving undifferentiated stem cells.

A Comparison of Neurosphere Differentiation Potential of Canine Bone Marrow-Derived Mesenchymal Stem Cells and Adipose-Derived Mesenchymal Stem Cells

Stem cell transplantation is one of the most promising yet enigmatic treatments for spinal cord injury (SCI), a common problem in dogs. As pre-differentiated mesenchymal stem cells (MSCs) can be expanded and differentiated into neurospheres in vitro, before being transplanted back, they may prove to be more beneficial for treating SCI. Therefore, we compared the endogenous differentiation potential, including the neuronal cell differentiation, of neurospheres from canine bone marrow MSCs (cBMMSCs) with that of the adipose tissue-derived MSCs (cADMSCs). Nestin-positive neurospheres were generated from MSCs derived from the bone marrow and adipose tissue. Neuronal cells were differentiated from the neurospheres derived from both these tissues. Gene expression analysis revealed that Nestin, βIII-tubulin, NCAM, OCT4 and SOX2 were expressed in MSCs and the corresponding neurospheres. Notably, cBMMSC-derived neuronal cells expressed higher levels of βIII-tubulin. The mRNA expressions of NANOG, Nestin, OCT4 and SOX2 were upregulated in neurospheres derived from both. Immunofluorescence analysis detected the expression of neuronal markers, namely, βIII-tubulin, GFAP, S100, NF200 and MAP2, in differentiated neuron-like cells. Our findings highlight that both cBMMSCs and cADMSCs could be differentiated into neurospheres and neuron-like cells, and therefore, these cells are suitable candidates for cell transplantation. Further, cADMSCs form a more suitable cell source, as larger number of cells could be harvested from cADMSC-derived neurospheres. Future studies employing in vivo transplantation models to investigate the effectiveness of MSCs for treating SCI are warranted.

The Therapeutic Effect of Bone Marrow-Derived Stem Cell Implantation After Epiphyseal Plate Injury Is Abrogated by Chondrogenic Predifferentiation

The goal of this study was to determine the effects of chondrogenic predifferentiation on the ability of bone marrow-derived stromal cells (BMSCs) delivered to growth plate defects to restore growth function. Chondrogenesis was induced with transforming growth factor (TGF)-β1 treatment in high-density monolayer cultures of BMSCs in vitro. The predifferentiated or undifferentiated BMSCs were either seeded into agarose gels for continued in vitro culture, or injected into growth plate defects via an in situ gelling agarose. Predifferentiated BMSCs had higher Sox-9, type II collagen, and aggrecan mRNA levels compared to undifferentiated cells after high-density monolayer culture. After transfer to agarose gels, predifferentiated cells did not produce a cartilaginous matrix, even with continued TGF-β1 stimulation, whereas undifferentiated cells produced a cartilaginous matrix in this system. Three-dimensional images of the growth plate created from microcomputed tomography scans showed that delivery of either predifferentiated or undifferentiated cells to defects resulted in a decrease in mineralized tether formation (fusion) in the growth plate tissue surrounding the defect to normal levels. Limb length discrepancy between injured and control limbs was corrected after treatment with undifferentiated, but not predifferentiated, cells. These results indicate that cell therapy may be an effective treatment to reduce growth dysfunction after growth plate injury, perhaps by maintaining the health of the uninjured growth plate tissue, and that the cell differentiation state plays a role in restoring the growth potential of the injured limb.

Implantation of undifferentiated and pre-differentiated human neural stem cells in the R6/2 transgenic mouse model of Huntington’s disease

BackgroundCell therapy is a potential therapeutic approach for several neurodegenetative disease, including Huntington Disease (HD). To evaluate the putative efficacy of cell therapy in HD, most studies have used excitotoxic animal models with only a few studies having been conducted in genetic animal models. Genetically modified animals should provide a more accurate representation of human HD, as they emulate the genetic basis of its etiology.ResultsIn this study, we aimed to assess the therapeutic potential of a human striatal neural stem cell line (STROC05) implanted in the R6/2 transgenic mouse model of HD. As DARPP-32 GABAergic output neurons are predominately lost in HD, STROC05 cells were also pre-differentiated using purmorphamine, a hedgehog agonist, to yield a greater number of DARPP-32 cells. A bilateral injection of 4.5x105 cells of either undifferentiated or pre-differentiated DARPP-32 cells, however, did not affect outcome compared to a vehicle control injection. Both survival and neuronal differentiation remained poor with a mean of only 161 and 81 cells surviving in the undifferentiated and differentiated conditions respectively. Only a few cells expressed the neuronal marker Fox3.ConclusionsAlthough the rapid brain atrophy and short life-span of the R6/2 model constitute adverse conditions to detect potentially delayed treatment effects, significant technical hurdles, such as poor cell survival and differentiation, were also sub-optimal. Further consideration of these aspects is therefore needed in more enduring transgenic HD models to provide a definite assessment of this cell line’s therapeutic relevance. However, a combination of treatments is likely needed to affect outcome in transgenic models of HD.

Effect of Adipose Tissue-Derived Osteogenic and Endothelial Cells on Bone Allograft Osteogenesis and Vascularization in Critical-Sized Calvarial Defects

The use of processed bone allograft to repair large osseous defects of the skull has been limited, given that it lacks the osteogenic cellularity and intrinsic vascular supply which are essential elements for successful graft healing and, at the same time, the areas to be targeted through tissue-engineering applications. In this study, we investigated the effect of predifferentiated rat adipose tissue-derived osteoblastic cells (OBs) and endothelial cells (ECs) on calvarial bone allograft healing and vascularization using an orthotopic critical-sized calvarial defect model. For this purpose, thirty-seven 8 mm critical calvarial defects in Lewis rats were treated with bone allografts seeded with no cells, undifferentiated adipose tissue-derived stem cells (ASC), OBs, ECs, and OBs and ECs simultaneously. After 8 weeks, the bone volume and mineral density were calculated using microcomputed tomography and the microvessel formation using immunohistochemical staining and imaging software. The amount of bone within the 8 mm defect was significantly higher for the allografts treated with ECs compared with the allografts treated with OBs (p=0.05) and simultaneously with the two cell lineages (p=0.02). There were no significant differences in bone formation between the latter two groups and the control groups (allografts treated with no cells and undifferentiated ASC). There were no significant differences in bone mineral density among the groups. The amount of microvessels was significantly higher in the group treated with ECs relative to all groups (p=< 0.05). Our results show that the implantation of ASC-derived ECs improves the vascularization of calvarial bone allografts at 8 weeks after treatment. This cell-based vascularization strategy can be used to improve the paucity of perfusion in allogenic bone implants. However, in this study, the treatment of allografts with OBs alone or in combination with ECs did not support bone formation or vascularization.

Functional recovery after spinal cord injury in dogs treated with a combination of Matrigel and neural-induced adipose-derived mesenchymal Stem cells

Previous studies have reported that scaffold or cell-based transplantation may improve functional recovery following spinal cord injury (SCI), but these results were based on neuronal regeneration and cell replacement. In this study, we investigated whether a combination of Matrigel and neural-induced mesenchymal stem cells (NMSC) improved hindlimb function in dogs with SCI, and what mechanisms were involved. We pre-differentiated canine adipose-derived mesenchymal stem cells into NMSC. A total of 12 dogs subjected to SCI procedures were assigned to one of the following three transplantation treatment groups: phosphate-buffered saline (PBS); Matrigel; or Matrigel seeded with NMSC. Treatment occurred 1 week after SCI. Basso, Beattie and Bresnahan (B.B.B.) and Tarlov scores, histopathology, immunofluorescence staining and Western blot analysis were used to evaluate the treatment effects. Compared with dogs administered PBS or Matrigel alone, dogs treated with Matrigel + NMSC showed significantly better functional recovery 8 weeks after transplantation. Histology and immunochemical analysis revealed that the combination of Matrigel + NMSC reduced fibrosis from secondary injury processes and improved neuronal regeneration more than the other treatments. In addition, the combination of Matrigel + NMSC decreased the expression of inflammation and/or astrogliosis markers. Increased expressions of intracellular molecules related to neuronal extension, neuronal markers and neurotrophic factors were also found in the Matrigel + NMSC group. However, the expression of nestin as a neural stem cell marker was increased with Matrigel alone. The combination of Matrigel + NMSC produced beneficial effects in dogs with regard to functional recovery following SCI through enhancement of anti-inflammation, anti-astrogliosis, neuronal extension and neuronal regeneration effects.

In Vitro Generation of Mature T Lymphocytes From Human Ips Cells and Genetic Analysis of TCR Gene Rearrangements

Abstract 2984T lymphocytes play central roles in cellular immunity, exerting their proliferative and effector activities when they recognize antigens via T-cell receptors (TCRs) in HLA-restricted and antigen-specific manner. Adoptive cell transfer therapy (ACT), the administration of ex vivo-activated and -expanded autologous tumor-reactive T lymphocytes, is currently one of the effective methods for immunotherapy, especially for treatment of metastatic solid tumors including melanoma. However, the successful applications of this method are currently limited for tumor therapies. To broaden the range of the application of ACT, we endeavored to develop easier method to obtain cells that carry antigen-specific TCR genes. For the purpose, generation of induced pluripotent stem (iPS) cells from an antigen-reactive single T lymphocyte is attractive and rewarding way. iPS cells have a capacity for unlimited self-renewal while maintaining pluripotency. These features may enable us to induce an unlimited number of T lymphocytes, especially high proliferative naïve / central memory-type T lymphocytes, showing reactivity to specific antigens. If they retain properties of naïve T lymphocytes, they may proliferate for a longer period and achieve better therapeutic effects than their peripheral blood counterparts expanded in vitro.Peripheral T lymphocytes were isolated from healthy volunteers. Then reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) were transduced into fresh or frozen / thawed T lymphocytes. T lymphocyte-derived iPS-like colonies were observed within 3 weeks and they were isolated and clonally expanded. They exhibited standard ES-like morphology, cell surface marker expression, alkaline phosphatase activity, as well as differentiation potential into various tissues related to all three germ layers. Human TCRs are encoded in four genes (TCRA, TCRB, TCRG, TCRD), which should be genetically assembled in an irreversible manner during T-lymphocyte development. This feature allowed us to retrospectively confirm that the iPS cells were generated from T lymphocyte. The TCR gene rearrangements encoded in an iPS colony were clonal for all iPS lines, indicating that each iPS colony was derived from a single T lymphocyte. Sequence analyses of TCR genes revealed whether the rearrangements were productive, and the productivity might promise the conservation of TCR genes rearrangement during the reprogramming process.Next, we tried to re-differentiate T lymphocyte derived-iPS (T-iPS) cells into T cells by co-culturing them with murine stromal cell layers (OP9 and OP9-DL1). T-cell differentiation was evidenced by the expression of T-cell markers, such as CD5, CD7, CD27, CD4, CD8, TCR α β and CD3. We obtained 33.5 ± 17.9% CD4+ CD8+ double positive (DP) cells, 6.51 ± 5.40% CD4+ CD8− single positive (SP) cells and 3.80 ± 1.28% CD4− CD8+ SP cells. They could be activated via TCR stimulation, and produce cytokines as functionally matured T lymphocytes do. The re-differentiation efficiency of T-iPS cells was higher than those of other pluripotent stem cells, such as embryonic stem (ES) cells, fibroblasts derived-iPS cells, or cord blood derived-iPS cells. Transcribed TCR mRNA sequences in re-differentiated T cells were analyzed, and they were revealed to be identical to that engraved in the pre-differentiated T-iPS cells genome in CD4+ CD8+ DP phase. However, fully matured CD4+ CD8− or CD4− CD8+ SP phase cells had several TCRA gene rearrangement patterns distinct from the original T-iPS cell’s. On the other hand, TCRB gene maintained identity with the original. The variance of the sequences, especially antigen-recognition site sequences, indicated that the antigen-specificity in the original T lymphocyte might be converted during DP to SP transition process in vitro.These data indicate that functionally matured T cells were generated by re-differentiating T-iPS cells in vitro, and that re-assemble of TCRA genes could take place during SP T cell maturation process. In order to fulfill the T-iPS-mediated immunotherapy, we need to overcome the obstacle of further TCRA gene rearrangements. We think the solution lies in refinement of the re-differentiation method for controlling the expression of RAG1 and RAG2 recombinases or for inhibiting their activities. Disclosures:No relevant conflicts of interest to declare.

Adipose-derived stem cells (ASCs) as a source of endothelial cells in the reconstruction of endothelialized skin equivalents

Tissue-engineered autologous skin is a potential alternative to autograft for burn coverage, but produces poor clinical responses such as unsatisfactory graft intake due to insufficient vascularization. Endothelialized skin equivalents comprising human umbilical vein endothelial cells (HUVECs) survive significantly longer due to inosculation with the capillaries of the host, but these cells are allogeneic by definition. The aim of this study was to reconstruct an autologous endothelialized skin equivalent by incorporating progenitor or pre-differentiated endothelial cells derived from adipose tissue, easily accessible source for autologous transplantation. Human adipose tissue-derived stem cells were isolated from lipoaspirates and amplified to obtain endothelial progenitor cells, which were subsequently differentiated into endothelial cells. These cells were then seeded along with human fibroblasts into a porous collagen-glycosaminoglycan-chitosan scaffold to obtain an endothelialized dermal equivalent. Then, human keratinocytes give rise to a endothelialized skin equivalent. Immunohistochemistry and transmission electron microscopy results demonstrate the presence of capillary-like tubular structures in skin equivalents comprising pre-differentiated endothelial cells, but not endothelial progenitor cells. The former expressed both EN4 and von Willebrand factor, and Weibel-Palade bodies were detected in their cytoplasm. This study demonstrates that adipose tissue is an excellent source of autologous endothelial cells to reconstruct endothelialized tissue equivalents, and that pre-differentiation of stem cells is necessary to obtain vasculature in such models.

Effects of Dibutyryl Cyclic-AMP on Survival and Neuronal Differentiation of Neural Stem/Progenitor Cells Transplanted into Spinal Cord Injured Rats

Neural stem/progenitor cells (NSPCs) have great potential as a cell replacement therapy for spinal cord injury. However, poor control over transplant cell differentiation and survival remain major obstacles. In this study, we asked whether dibutyryl cyclic-AMP (dbcAMP), which was shown to induce up to 85% in vitro differentiation of NSPCs into neurons would enhance survival of transplanted NSPCs through prolonged exposure either in vitro or in vivo through the controlled release of dbcAMP encapsulated within poly(lactic-co-glycolic acid) (PLGA) microspheres and embedded within chitosan guidance channels. NSPCs, seeded in fibrin scaffolds within the channels, differentiated in vitro to betaIII-tubulin positive neurons by immunostaining and mRNA expression, in response to dbcAMP released from PLGA microspheres. After transplantation in spinal cord injured rats, the survival and differentiation of NSPCs was evaluated. Untreated NSPCs, NSPCs transplanted with dbcAMP-releasing microspheres, and NSPCs pre-differentiated with dbcAMP for 4 days in vitro were transplanted after rat spinal cord transection and assessed 2 and 6 weeks later. Interestingly, NSPC survival was highest in the dbcAMP pre-treated group, having approximately 80% survival at both time points, which is remarkable given that stem cell transplantation often results in less than 1% survival at similar times. Importantly, dbcAMP pre-treatment also resulted in the greatest number of in vivo NSPCs differentiated into neurons (37±4%), followed by dbcAMP-microsphere treated NSPCs (27±14%) and untreated NSPCs (15±7%). The reverse trend was observed for NSPC-derived oligodendrocytes and astrocytes, with these populations being highest in untreated NSPCs. This combination strategy of stem cell-loaded chitosan channels implanted in a fully transected spinal cord resulted in extensive axonal regeneration into the injury site, with improved functional recovery after 6 weeks in animals implanted with pre-differentiated stem cells in chitosan channels.

Membrane Culture of Bone Marrow Stromal Cells Yields Better Tissue Than Pellet Culture for Engineering Cartilage-Bone Substitute Biphasic Constructs in a Two-Step Process

Our long-term goal is to treat osteochondral lesions with bioengineered biphasic constructs. We have previously demonstrated that biphasic constructs, created in vitro with primary chondrocytes harvested from healthy joints and a porous calcium polyphosphate (CPP) substrate bone substitute, could successfully repair a focal defect in sheep joints. However, primary chondrocytes are limited in supply and cannot be used in engineering constructs large enough for clinical use. Thus, we developed a robust protocol to predifferentiate sheep bone marrow-derived stromal cells to chondrocytes on collagen-coated polytetrafluoroethane membrane inserts, and harvest the chondrocytes that develop and subsequently culturing these predifferentiated cells scaffold-free on the intended articulation surface of the CPP. Chondrocytes predifferentiated on membrane culture accumulated similar matrix as those in conventional pellet culture, but expressed less Col1a1 RNA. Membrane culture predifferentiated cells gave rise to a functionally superior hyaline cartilage tissue compared to pellet culture predifferentiated cells. Studies demonstrated that 2 weeks of membrane predifferentiation culture followed by 8 weeks of biphasic construct culture was the optimal culture period at which the compressive mechanical strength and the accumulation of extracellular matrix were maximized while avoiding tissue mineralization. This protocol will be used to generate implants for preclinical study to determine their ability to repair osteochondral lesions.

Transplantation of predifferentiated adipose-derived stromal cells for the treatment of spinal cord injury.

Adipose-derived stromal cells (ASCs) are an alternative source of stem cells for cell-based therapies of neurological disorders such as spinal cord injury (SCI). In the present study, we predifferentiated ASCs (pASCs) and compared their behavior with naïve ASCs in vitro and after transplantation into rats with a balloon-induced compression lesion. ASCs were predifferentiated into spheres before transplantation, then pASCs or ASCs were injected intraspinally 1 week after SCI. The cells' fate and the rats' functional outcome were assessed using behavioral, histological, and electrophysiological methods. Immunohistological analysis of pASCs in vitro revealed the expression of NCAM, NG2, S100, and p75. Quantitative RT-PCR at different intervals after neural induction showed the up-regulated expression of the glial markers NG2 and p75 and the neural precursor markers NCAM and Nestin. Patch clamp analysis of pASCs revealed three different types of membrane currents; however, none were fast activating Na(+) currents indicating a mature neuronal phenotype. Significant improvement in both the pASC and ASC transplanted groups was observed in the BBB motor test. In vivo, pASCs survived better than ASCs did and interacted closely with the host tissue, wrapping host axons and oligodendrocytes. Some transplanted cells were NG2- or CD31-positive, but no neuronal markers were detected. The predifferentiation of ASCs plays a beneficial role in SCI repair by promoting the protection of denuded axons; however, functional improvements were comparable in both the groups, indicating that repair was induced mainly through paracrine mechanisms.