Adult Human Mesenchymal Stem Cells Research Articles

Long-Term Effects of Adipose-Derived Stem Cells for the Treatment of Bilateral Limbal Stem Cell Deficiency

Purpose To determine the safety and feasibility of human autologous adipose tissue-derived adult mesenchymal stem cells (ASCs) for ocular surface regeneration in patients with bilateral limbal stem-cell deficiency (LSCD). Methods A phase IIa clinical trial was designed (https://Clinicaltrials.gov, NCT01808378) with 8 patients, 3 of whom had aniridia, 2 meibomian glands diseases, 2 multiple surgeries and 1 chronic chemical injury. The therapeutic protocol was as follows: 6-mm of central corneal epithelium was removed, 400,000 ASCs were injected into each limboconjunctival quadrant, 400,000 ASCs were suspended over the cornea for 20 min, and finally the cornea was covered with an amniotic membrane patch. Results No adverse events were detected after a mean of 86,5 months of follow-up. One year after surgery, 6 of the 8 transplants were scored as successful, five patients had improved uncorrected visual acuity (mean of 12 letters), two patients presented epithelial defects (also present at baseline) and the mean percentage of corneal neovascularization was of 28.75% (36.98%, at baseline). Re-examination 24 months after treatment disclosed preserved efficacy in 4 patients. At the last visit (after a mean of 86,5 months of follow up) epithelial defects were absent in all patients although improvement in all of the variables was only maintained in patient 3 (meibomian glands agenesia). Conclusion ASCs are a feasible and conservative therapy for treating bilateral LSCD. The therapeutic effect differs between etiologies and diminishes over time.

Surface-Functionalized Stem Cell-Derived Extracellular Vesicles for Vascular Elastic Matrix Regenerative Repair.

Extracellular vesicles (EVs) are nanosized vesicles that carry cell-specific biomolecular information. Our previous studies showed that adult human bone marrow mesenchymal stem cell (BM-MSC)-derived EVs provide antiproteolytic and proregenerative effects in cultures of smooth muscle cells (SMCs) derived from an elastase-infused rat abdominal aortic aneurysm (AAA) model, and this is promising toward their use as a therapeutic platform for naturally irreversible elastic matrix aberrations in the aortic wall. Since systemically administered EVs poorly home into sites of tissue injury, disease strategies to improve their affinity toward target tissues are of great significance for EV-based treatment strategies. Toward this goal, in this work, we developed a postisolation surface modification strategy to target MSC-derived EVs to the AAA wall. The EVs were surface-conjugated with a short, synthetic, azide-modified peptide sequence for targeted binding to cathepsin K (CatK), a cysteine protease overexpressed in the AAA wall. Conjugation was performed using a copper-free click chemistry method. We determined that such conjugation improved EV uptake into cultured aneurysmal SMCs in culture and their binding to the wall of matrix injured vessels ex vivo. The proregenerative and antiproteolytic effects of MSC-EVs on cultured rat aneurysmal SMCs were also unaffected following peptide conjugation. From this study, it appears that modification with short synthetic peptide sequences seems to be an effective strategy for improving the cell-specific uptake of EVs and may be effective in facilitating AAA-targeted therapy.

Derivation of Limbal Stem Cells from Human Adult Mesenchymal Stem Cells for the Treatment of Limbal Stem Cell Deficiency.

Approximately 10 million individuals have blindness due to limbal stem cell (LSCs) deficiency, one of the most challenging problems in ophthalmology. To replenish the LSC pool, an autologous extraocular cell source is appropriate, thereby avoiding the risk of immune rejection, the need for immunosuppression and the risk of damaging the contralateral eye. In recent years, adipose-derived mesenchymal stem cells (ADSCs) have been a key element in ocular regenerative medicine. In this study, we developed a protocol for deriving human LSCs from ADSCs compatible with the standard carrier human amniotic membrane, helping provide a stem cell pool capable of maintaining proper corneal epithelial homeostasis. The best protocol included an ectodermal induction step by culturing ADSCs with media containing fetal bovine serum, transforming growth factor-β inhibitor SB-505124, Wnt inhibitor IWP-2 and FGF2 for 7 days, followed by an LSC induction step of culture in modified supplemental hormonal epithelial medium supplemented with pigment epithelium-derived factor and keratinocyte growth factor for 10 additional days. The optimal differentiation efficiency was achieved when cells were cultured in this manner over vitronectin coating, resulting in up to 50% double-positive αp63/BMI-1 cells. The results of this project will benefit patients with LSC deficiency, aiding the restoration of vision.

Novel Drugs in the Treatment of Steroid Refractory Acute Graft Versus Host Disease

Steroid refractory acute graft versus host disease (SR-aGVHD) remains an area of unmet needs with more therapeutic options required. Here, we review novel treatment options for SR-aGVHD. Methods: A comprehensive data search was done across various data sets, including PubMed, Cochrane, and Embase, using MeSH terms and keywords to look for all the different drug types that have been studied for SR-aGVHD. A summary of the results is described here. Results: Denilekin diftitox (Ontak), a recombinant protein composed of IL-2 fused to diphtheria toxin, was studied in a phase 1 trial by Vincent et al. Thirty patients receiving 9 g/kg of Ontak at different intervals showed a Complete response (CR) of 38% and a partial response (PR) of 38%. Remetemcel-L, ex vivo cultured adult human mesenchymal stem cells were studied in a multicenter phase III trial in 260 patients by Kebraei et al. compared to placebo. At a dose of 2 x 106 hMSC/kg, an overall response rate (ORR) of 58% vs. 54% in the placebo group was seen with CR of 35 and 30% respectively at day 28. Third-party bone marrow-derived mesenchymal stromal cells (MSC) expanded in platelet lysate were studied by Introna et al. in a phase 1 trial. Forty patients received a median dose of 1.5 x 10(6)/kg per infusion and showed an ORR of 67.5% with a CR of 27.5%. OS at 1 and 2 years was 50% and 38.6%, respectively. Pentostatin, an adenosine deaminase inhibitor, was studied by Meade et al. in a phase 1 trial in 23 patients. Pentostatin was administered at a dose of 1 to 4 mg/m2/day. Study showed a CR of 63%. Sirolimus, an mTOR Inhibitor, was studied by Benito et al. in a pilot study in 21 patients. On an average dose of 5 mg/m2, sirolimus showed a CR of 23% and PR of 33%. Couriel et al., in a phase III trial, 63 patients were randomized and received either methylprednisone (MP) 2mg/kg or infliximab plus MP. On days 7 and 28 response rate for infliximab + MP vs. MP was 52% vs. 78% and 62% vs. 58%, respectively. Przepiorka et al. reported the results of an open-label phase II study in 20 patients with SR-aGVHD treated with BTI-322 (LO-CD2a), an anti-human CD2 SR-aGVHD IgG2b monoclonal antibody (MAB). ORR was achieved in 55% of the patients. Deeg et al. resulted from a dose-escalation study in 59 patients with steroid-refractory aGVHD treated with ABX-CBL MAB. Reported ORR was 51%. Carpent et al. reported the results of a phase II trial in which 44 patients were treated with visilizumab with ORR of 32% at day 42. Bordigoni et al. treated 62 patients with daclizumab in a phase II trial with reportef ORR of 68.80% at 44 months follow up. Groth et al. studied anti-CD3, anti CD7 antibodies separately conjugated to recombinant ricin A (CD3/CD7-1 T) in adults, 18 years and older with grade III to IV aGVHD following HSCT for myeloid and lymphoid malignancies. Twenty patients were treated with an ORR of 60% on day 28. Adverse events are listed in the Table 2. Conclusion Different targeted drugs focused on the specific pathways show promising results. Monoclonal antibodies in particular showed good treatment outcomes with tolerable safety profile. Further studies are required to identify a valuable therapeutic option, SR-aGVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cell-Laden Composite Hydrogel Bioinks with Human Bone Allograft Particles to Enhance Stem Cell Osteogenesis.

There is a growing demand for bone graft substitutes that mimic the extracellular matrix properties of the native bone tissue to enhance stem cell osteogenesis. Composite hydrogels containing human bone allograft particles are particularly interesting due to inherent bioactivity of the allograft tissue. Here, we report a novel photocurable composite hydrogel bioink for bone tissue engineering. Our composite bioink is formulated by incorporating human allograft bone particles in a methacrylated alginate formulation to enhance adult human mesenchymal stem cell (hMSC) osteogenesis. Detailed rheology and printability studies confirm suitability of our composite bioinks for extrusion-based 3D bioprinting technology. In vitro studies reveal high cell viability (~90%) for hMSCs up to 28 days of culture within 3D bioprinted composite scaffolds. When cultured within bioprinted composite scaffolds, hMSCs show significantly enhanced osteogenic differentiation as compared to neat scaffolds based on alkaline phosphatase activity, calcium deposition, and osteocalcin expression.

Modeling early changes associated with cartilage trauma using human-cell-laden hydrogel cartilage models

BackgroundTraumatic impacts to the articular joint surface are known to lead to cartilage degeneration, as in post-traumatic osteoarthritis (PTOA). Limited progress in the development of disease-modifying OA drugs (DMOADs) may be due to insufficient mechanistic understanding of human disease onset/progression and insufficient in vitro models for disease and therapeutic modeling. In this study, biomimetic hydrogels laden with adult human mesenchymal stromal cells (MSC) are used to examine the effects of traumatic impacts as a model of PTOA. We hypothesize that MSC-based, engineered cartilage models will respond to traumatic impacts in a manner congruent with early PTOA pathogenesis observed in animal models.MethodsEngineered cartilage constructs were fabricated by encapsulating adult human bone marrow-derived mesenchymal stem cells in a photocross-linkable, biomimetic hydrogel of 15% methacrylated gelatin and promoting chondrogenic differentiation for 28 days in a defined medium and TGF-β3. Constructs were subjected to traumatic impacts with different strains or 10 ng/ml IL-1β, as a common comparative method of modeling OA. Cell viability and metabolism, elastic modulus, gene expression, matrix protein production and activation of catabolic enzymes were assessed.ResultsCell viability staining showed that traumatic impacts of 30% strain caused an appropriate level of cell death in engineered cartilage constructs. Gene expression and histo/immunohistochemical analyses revealed an acute decrease in anabolic activities, such as COL2 and ACAN expression, and a rapid increase in catabolic enzyme expression, e.g., MMP13, and inflammatory modulators, e.g., COX2. Safranin O staining and GAG assays together revealed a transient decrease in matrix production 24 h after trauma that recovered within 7 days. The decrease in elastic modulus of engineered cartilage constructs was coincident with GAG loss and mediated by the encapsulated cells. The acute and transient changes observed after traumatic impacts contrasted with progressive changes observed using continual IL-1β treatment.ConclusionsTraumatic impacts delivered to engineered cartilage constructs induced PTOA-like changes in the encapsulated cells. While IL-1b may be appropriate in modeling OA pathogenesis, the results of this study indicate it may not be appropriate in understanding the etiology of PTOA. The development of a more physiological in vitro PTOA model may contribute to the more rapid development of DMOADs.

Multinuclear MRI Reveals Early Efficacy of Stem Cell Therapy in Stroke.

Compromised adult human mesenchymal stem cells (hMSC) can impair cell therapy efficacy and further reverse ischemic recovery. However, in vitro assays require extended passage to characterize cells, limiting rapid assessment for therapeutic potency. Multinuclear magnetic resonance imaging and spectroscopy (MRI/S) provides near real-time feedback on disease progression and tissue recovery. Applied to ischemic stroke, 23Na MRI evaluates treatment efficacy within 24h after middle cerebral artery occlusion, showing recovery of sodium homeostasis and lesion reduction in specimens treated with hMSC while 1H MRS identifies reduction in lactate levels. This combined metric was confirmed by evaluating treatment groups receiving healthy or compromised hMSC versus vehicle (sham saline injection) over 21days. Behavioral tests to assess functional recovery and cell analysis for immunomodulatory and macrophage activity to detect hMSC potency confirm MR findings. Clinically, these MR metrics may prove critical to early evaluations of therapeutic efficacy and overall stroke recovery.

Nitrogen-doped titanium dioxide films fabricated via magnetron sputtering for vascular stent biocompatibility improvement

Nowadays, vascular stents are commonly used to treat cardiovascular diseases. This article focuses on the influence of nitrogen doping of titanium dioxide thin films, utilized for coating metallic stents to improve their biological properties and biocompatibility. The hereby-investigated titanium oxide thin films are fabricated by magnetron sputtering in a reactive gas atmosphere consisting of argon and oxygen in the first case and argon, nitrogen and oxygen in the second case. Control of the nitrogen and oxygen gas flow rates, and hence their mixing ratios, allows adjustment of the nitrogen-doping level within the titanium dioxide thin films. A correlation of the thin film internal structure on the in vitro behavior of human mesenchymal stem cells derived from adipose tissue is hereby demonstrated. Different nitrogen doping levels affect the surface energy, the wettability, the cell adhesion and thus the cellular proliferation on top of the thin films. The surface colonization of cells on titanium dioxide thin films decreases up to a nitrogen-doping level of ∼ 3.75 at.%, which is associated with a decreasing polar component of the surface energy. For non-doped titanium dioxide thin films, a weak chondrogenesis of adult human adipose-derived mesenchymal stem cells with lower chondrogenic differentiation compared to glass is observed. An increasing nitrogen-doping level leads to linear increase in the chondrogenic differentiation rate, which is comparable to the control value of uncoated glass. Other investigated differentiated cell types do not display this behavior.

Suitability of Human Mesenchymal Stem Cells Derived from Fetal Umbilical Cord (Wharton's Jelly) as an Alternative In Vitro Model for Acute Drug Toxicity Screening.

Preclinical toxicity screening is the first and most crucial test that assesses the safety of new candidate drugs before their consideration for further evaluation in clinical trials. In vitro drug screening using stem cells has lately arisen as a promising alternative to the “gold standard” of animal testing, but their suitability and performance characteristics in toxicological studies have so far not been comprehensively investigated. In this study, we focused on the evaluation of human mesenchymal stem cells isolated from the matrix (Wharton’s jelly) of fetal umbilical cord (WJSCs), which bear enhanced in vitro applicability due to their unique biological characteristics. In order to determine their suitability for drug-related cytotoxicity assessment, we adopted a high-throughput methodology that evaluated their sensitivity to a selected panel of chemicals in different culture environments. Cytotoxicity was measured within 48 h by means of MTS and/or NRU viability assays, and was compared directly (in vitro) or indirectly (in silico) to adult human mesenchymal stem cells and to reference cell lines of human and murine origin. Our data clearly suggest that human WJSCs can serve as a robust in vitro alternative for acute drug toxicity screening by uniquely combining rapid and versatile assay setup with high-throughput analysis, good representation of human toxicology, high reproducibility, and low cost.

WNT/beta-catenin signalling interrupts a senescence-induction cascade in human mesenchymal stem cells that restricts their expansion

Senescence, the irreversible cell cycle arrest of damaged cells, is accompanied by a deleterious pro-inflammatory senescence-associated secretory phenotype (SASP). Senescence and the SASP are major factors in aging, cancer, and degenerative diseases, and interfere with the expansion of adult cells in vitro, yet little is known about how to counteract their induction and deleterious effects. Paracrine signals are increasingly recognized as important senescence triggers and understanding their regulation and mode of action may provide novel opportunities to reduce senescence-induced inflammation and improve cell-based therapies. Here, we show that the signalling protein WNT3A counteracts the induction of paracrine senescence in cultured human adult mesenchymal stem cells (MSCs). We find that entry into senescence in a small subpopulation of MSCs triggers a secretome that causes a feed-forward signalling cascade that with increasing speed induces healthy cells into senescence. WNT signals interrupt this cascade by repressing cytokines that mediate this induction of senescence. Inhibition of those mediators by interference with NF-κB or interleukin 6 signalling reduced paracrine senescence in absence of WNT3A and promoted the expansion of MSCs. Our work reveals how WNT signals can antagonize senescence and has relevance not only for expansion of adult cells but can also provide new insights into senescence-associated inflammatory and degenerative diseases.

Improvement of an Effective Protocol for Directed Differentiation of Human Adipose Tissue-Derived Adult Mesenchymal Stem Cells to Corneal Endothelial Cells.

Corneal disease affects 12.5 million individuals worldwide, with 2 million new cases each year. The standard treatment consists of a corneal transplantation from a human donor; however, the worldwide demand significantly exceeds the available supply. Lamellar endothelial keratoplasty, the replacement of only the endothelial layer of the cornea, can partially solve the problem. Progressive efforts have succeeded in expanding hCECs; however, the ability to expand hCECs is still limited, and new sources of CECs are being sought. Crucial advances have been achieved by the directed differentiation of embryonic or induced pluripotent stem cells, but these cells have disadvantages, such as the use of oncogenes, and are still difficult to establish. We aimed to transfer such knowledge to obtain hCECs from adipose tissue-derived adult mesenchymal stem cells (ADSC) by modifying four previously published procedures. We present several protocols capable of the directed differentiation of human ADSCs to hCECs. In our hands, the protocol by Ali et al. was the best adapted to such differentiation in terms of efficiency, time, and financial cost; however, the protocol by Wagoner et al. was the best for CEC marker expression. Our results broaden the type of cells of autologous extraocular origin that could be employed in the clinical setting for corneal endothelial deficiency.

Modifications in Gene Expression in the Process of Osteoblastic Differentiation of Multipotent Bone Marrow-Derived Human Mesenchymal Stem Cells Induced by a Novel Osteoinductive Porous Medical-Grade 3D-Printed Poly(ε-caprolactone)/β-tricalcium Phosphate Composite.

In this work, we evaluated the influence of a novel hybrid 3D-printed porous composite scaffold based on poly(ε-caprolactone) (PCL) and β-tricalcium phosphate (β-TCP) microparticles in the process of adhesion, proliferation, and osteoblastic differentiation of multipotent adult human bone marrow mesenchymal stem cells (ah-BM-MSCs) cultured under basal and osteogenic conditions. The in vitro biological response of ah-BM-MSCs seeded on the scaffolds was evaluated in terms of cytotoxicity, adhesion, and proliferation (AlamarBlue Assay®) after 1, 3, 7, and 14 days of culture. The osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, mineralization (Alizarin Red Solution, ARS), expression of surface markers (CD73, CD90, and CD105), and reverse transcription–quantitative polymerase chain reaction (qRT-PCR) after 7 and 14 days of culture. The scaffolds tested were found to be bioactive and biocompatible, as demonstrated by their effects on cytotoxicity (viability) and extracellular matrix production. The mineralization and ALP assays revealed that osteogenic differentiation increased in the presence of PCL/β-TCP scaffolds. The latter was also confirmed by the gene expression levels of the proteins involved in the ossification process. Our results suggest that similar bio-inspired hybrid composite materials would be excellent candidates for osteoinductive and osteogenic medical-grade scaffolds to support cell proliferation and differentiation for tissue engineering, which warrants future in vivo research.

An implantable human stem cell-derived tissue-engineered rostral migratory stream for directed neuronal replacement

The rostral migratory stream (RMS) facilitates neuroblast migration from the subventricular zone to the olfactory bulb throughout adulthood. Brain lesions attract neuroblast migration out of the RMS, but resultant regeneration is insufficient. Increasing neuroblast migration into lesions has improved recovery in rodent studies. We previously developed techniques for fabricating an astrocyte-based Tissue-Engineered RMS (TE-RMS) intended to redirect endogenous neuroblasts into distal brain lesions for sustained neuronal replacement. Here, we demonstrate that astrocyte-like-cells can be derived from adult human gingiva mesenchymal stem cells and used for TE-RMS fabrication. We report that key proteins enriched in the RMS are enriched in TE-RMSs. Furthermore, the human TE-RMS facilitates directed migration of immature neurons in vitro. Finally, human TE-RMSs implanted in athymic rat brains redirect migration of neuroblasts out of the endogenous RMS. By emulating the brain’s most efficient means for directing neuroblast migration, the TE-RMS offers a promising new approach to neuroregenerative medicine.

Matrigel enhances differentiation of human adipose tissue-derived stem cells into dopaminergic neuron

BackgroundTherapy based stem cells have offered a novel therapeutic approach for the improvement of neurodegenerative diseases, specially Parkinson. Hence, developing a well-established culture model with appropriate stem cells is extremely crucial in regenerative engineering to provide efficient targeted cells. Human adult mesenchymal stem cells derived from adipose tissue (hADSCs) have emerged as a promising source of stem cells due to their unique potentials of self-renewal and differentiation into other stem cells. The purpose of this study was to investigate the differentiation capacity of hADSCs into dopaminergic and neuron-like cells in the 3D culture plate (Matrigel). Methods and materialshADSCs were obtained from adipose tissues of patients and then characterized morphologically with flowcytometry. Isolated cells were harvested to perform differentiation on Matrigel and tissue culture plate (TCP) supplemented with induction factors. The survival rate of cells during neural induction was monitored by MTT. The expression of specific cell markers was analyzed by QRT-PCR and immunocytochemistry on days 2, 8 and 14. The level of released dopamine was measured using HPLC technique. ResultsMatrigel had a positive effect on maintaining cell growth compared to those on TCP. Moreover, the number of TH and MAPII positive cells is substantially higher in Matrigel than in TCP. Sox2 and Nestin had a prominent expression in hADSCs within the first days of differentiation. The gene expression of neural markers such as TH, Nurr1, LMX1A and DAT was detected and increased after day 8. Moreover, the dopamine released in the cell harvested on Matrigel was greater than those seeded on TCP. ConclusionsOverall, hADSCs could generate dopaminergic cells, which suggest its strong capability to serve as a tool for Parkinson disease model in the regenerative medicine.

BPA and BPA alternatives BPS, BPAF, and TMBPF, induce cytotoxicity and apoptosis in rat and human stem cells

Bisphenol A (BPA) is a ubiquitous industrial chemical found in everyday plastic products and materials. Due to scientific findings on the reproductive, developmental, and cellular defects caused by BPA and heightened public awareness, manufacturers have begun to use new chemicals in place of BPA in “BPA-free” products. These alternatives are chemical analogs of BPA and include dozens of new compounds that have undergone relatively little testing and oversight, including: bisphenol S (BPS), bisphenol AF (BPAF), and the recently developed tetramethyl bisphenol F (TMBPF; the monomer of valPure V70). Here, we used adult female rat adipose-derived stem cells (rASCs) and human mesenchymal stem cells (hMSCs) to compare the toxicities and potencies of these BPA alternatives in vitro. Rat and human stem cells were exposed to BPA (1–10 μM), 17β-estradiol (E2; 10 μM), BPS (1–100 μM), BPAF (3×10−4–30 μM), TMBPF (0.01–50 μM), or control media alone (with 0.01% ethanol) for varying time intervals from 10 min to 24 h. We found significantly decreased cell viability and massive apoptosis in rat and human stem cells treated with each BPA analog, as early as 10 min of exposure, and at low, physiologically relevant doses. BPAF showed extreme cytotoxicity in a dose-dependent manner (LC50 =0.014 μM (rASCs) and 0.009 μM (hMSCs)), whereas TMBPF showed a bimodal response, with low and high concentrations being the most toxic (LC50 =0.88 μM (rASCs) and 0.06 μM (hMSCs)). Activated caspase-6 levels increased in nearly all cells treated with the BPA analogs indicating the majority of cell death was due to caspase-6-mediated apoptosis. These results in both rat and human stem cells underscore the toxicity and potency of these BPA analogs, and establish a rank order of potency of: BPAF>TMBPF>BPA>BPS. Further, these and other recent findings indicate that these newer BPA analogs may be ‘regrettable substitutions,’ being worse than the original parent compound and lacking proper testing and regulation. This work brings to light the need for further toxicological characterization, better regulation, greater public awareness, and the development of safer, more sustainable chemicals and non-plastic products.

Micro-/Nano-Structured Ceramic Scaffolds That Mimic Natural Cancellous Bone.

Micro-/nano-structured scaffolds with a weight composition of 46.6% α-tricalcium phosphate (α-TCP)—53.4% silicocarnotite (SC) were synthesized by the polymer replica method. The scanning electron microscopy (SEM) analysis of the scaffolds and natural cancellous bone was performed for comparison purposes. Scaffolds were obtained at three cooling rates via the eutectoid temperature (50 °C/h, 16.5 °C/h, 5.5 °C/h), which allowed the surface nanostructure and mechanical strength to be controlled. Surface nanostructures were characterized by transmission electron microscopy (TEM) and Raman analysis. Both phases α-TCP and SC present in the scaffolds were well-identified, looked compact and dense, and had neither porosities nor cracks. The non-cytotoxic effect was evaluated in vitro by the proliferation ability of adult human mesenchymal stem cells (ah-MSCs) seeded on scaffold surfaces. There was no evidence for cytotoxicity and the number of cells increased with culture time. A dense cell-hydroxyapatite layer formed until 28 days. The SEM analysis suggested cell-mediated extracellular matrix formation. Finally, scaffolds were functionalized with the alkaline phosphatase enzyme (ALP) to achieve biological functionalization. The ALP was successfully grafted onto scaffolds, whose enzymatic activity was maintained. Scaffolds mimicked the micro-/nano-structure and chemical composition of natural cancellous bone by considering cell biology and biomolecule functionalization.

Application of a Hyperelastic 3D Printed Scaffold for Mesenchymal Stem Cell-Based Fabrication of a Bizonal Tendon Enthesis-like Construct

After surgical tendon repair, the tendon-to-bone enthesis often does not regenerate, which leads to high numbers of rupture recurrences. To remedy this, tissue engineering techniques are being pursued to strengthen the interface and improve regeneration. In this study, we used hyperelastic biphasic 3D printed PLGA scaffolds with aligned pores at the tendon side and random pores at the bone side to mimic the native insertion side. In an attempt to recreate the enthesis, the scaffolds were seeded with adult human mesenchymal stem cells and then cultured in dual fluidic bioreactors, which allows the separate in-flow of tenogenic and chondrogenic differentiation media. MTS assay confirmed the ability of cells to proliferate in dual-flow bioreactors at similar levels to tissue culture plate. Hematoxylin-eosin staining confirmed a compact cell layer entrapped within newly deposited extracellular matrix attached to the scaffolds’ fibers and between the porous cavities, that increased with culture time. After 7, 14, and 21 days, samples were collected and analyzed by qRT-PCR and GAG production. Cultured constructs in dual fluidic bioreactors differentiate regionally toward a tenogenic or chondrogenic fate dependent on exposure to the corresponding differentiation medium. SOX9 gene expression was upregulated (up to 50-fold compared to control) in both compartments, with a more marked upregulation in the cartilaginous portion of the scaffold, By day 21, the cartilage matrix marker, collage II, and the tendon specific marker, tenomodulin, were found to be highly upregulated in the cartilaginous and tendinous portions of the construct, respectively. In addition, GAG production in the treated constructs (serum-free) matched that in control constructs exposed to 10% fetal bovine serum, confirming the support of functional matrix formation in this system. In summary, our findings have validated this dual fluidic system as a potential platform to form the tendon enthesis, and will be optimized in future studies to achieve the fabrication of distinctly biphasic constructs.

Characterization of traumatized muscle-derived multipotent progenitor cells from low-energy trauma

BackgroundMultipotent progenitor cells have been harvested from different human tissues, including the bone marrow, adipose tissue, and umbilical cord blood. Previously, we identified a population of mesenchymal progenitor cells (MPCs) isolated from the traumatized muscle of patients undergoing reconstructive surgery following a war-related blast injury. These cells demonstrated the ability to differentiate into multiple mesenchymal lineages. While distal radius fractures from a civilian setting have a much lower injury mechanism (low-energy trauma), we hypothesized that debrided traumatized muscle near the fracture site would contain multipotent progenitor cells with the ability to differentiate and regenerate the injured tissue.MethodsThe traumatized muscle was debrided from the pronator quadratus in patients undergoing open reduction and internal fixation for a distal radius fracture at the Walter Reed National Military Medical Center. Using a previously described protocol for the isolation of MPCs from war-related extremity injuries, cells were harvested from the low-energy traumatized muscle samples and expanded in culture. Isolated cells were characterized by flow cytometry and q-RT-PCRs and induced to adipogenic, osteogenic, and chondrogenic differentiation. Downstream analyses consisted of lineage-specific staining and q-RT-PCR.ResultsCells isolated from low-energy traumatized muscle samples were CD73+, CD90+, and CD105+ that are the characteristic of adult human mesenchymal stem cells. These cells expressed high levels of the stem cell markers OCT4 and NANOG 1-day after isolation, which was dramatically reduced over-time in monolayer culture. Following induction, lineage-specific markers were demonstrated by each specific staining and confirmed by gene expression analysis, demonstrating the ability of these cells to differentiate into adipogenic, osteogenic, and chondrogenic lineages.ConclusionsAdult multipotent progenitor cells are an essential component for the success of regenerative medicine efforts. While MPCs have been isolated and characterized from severely traumatized muscle from high-energy injuries, here, we report that cells with similar characteristics and multipotential capacity have been isolated from the tissue that was exposed to low-energy, community trauma.

Study of Mesenchymal Stem Cell-Mediated Mitochondrial Transfer in In Vitro Models of Oxidant-Mediated Airway Epithelial and Smooth Muscle Cell Injury.

Mesenchymal stem cells (MSCs) have emerged as an attractive candidate for cell-based therapy. In the past decade, many animal and pilot clinical studies have demonstrated that MSCs are therapeutically beneficial for the treatment of obstructive lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). However, due to the scarcity of adult human MSCs, human-induced pluripotent stem cells mesenchymal stem cells (iPSCs) are now increasingly used as a source of MSCs. iPSCs are derived by reprogramming somatic cells from a wide variety of tissues such as skin biopsies and then differentiating them into iPSC-MSCs. One of the mechanisms through which MSCs exert their protective effects is mitochondrial transfer. Specifically, transfer of mitochondria from iPSC-MSCs to lung cells was shown to protect lung cells against oxidative stress-induced mitochondrial dysfunction and apoptosis and to reduce lung injury and inflammation in in vivo models of lung disease. In this chapter, we detail our methods to visualize and quantify iPSC-MSC-mediated mitochondrial transfer and to study its effects on oxidant-induced airway epithelial and smooth muscle cell models of acute airway cell injury.

Formation of alginate microspheres prepared by optimized microfluidics parameters for high encapsulation of bioactive molecules

Drug delivery systems such as microspheres have shown potential in releasing biologicals effectively for tissue engineering applications. Microfluidic systems are especially attractive for generating microspheres as they produce microspheres of controlled-size and in low volumes, using micro-emulsion processes. However, the flow rate dependency on the encapsulation of molecules at a microscale is poorly understood. In particular, the flow rate and pressure parameters might influence the droplet formation and drug encapsulation efficiency. We evaluated the parameters within a two-reagent flow focusing microfluidic chip under continuous formation of hydrogel particles using a flourinated oil and an ionic crosslinkable alginate hydrogel. Fluorescein isothiocyanate-dextran sulfate (FITC-dextran sulfate MW: 40 kDa) was used to evaluate the variation of the encapsulation efficiency with the flow parameters, optimizing droplets and microsphere formation. The ideal flow rates allowing for maximum encapsulation efficiency, were utilised to form bioactive microspheres by delivering transforming growth factor beta-3 (TGFβ-3) in cell culture media. Finally, we evaluated the potential of microfluidic-formed microspheres to be included within biological environments. The biocompatibility of the microspheres was tested over 28 days using adult human mesenchymal stem cells (hMSCs). The release profile of the growth factors from microspheres showed a sustained release in media, after an initial burst, up to 30 days. The metabolic activity of the cells cultured in the presence of the microspheres was similar to controls, supporting the biocompatibility of this approach. The fine-tuned parameters for alginate hydrogel to form microspheres have potential in encapsulating and preserving functional structure of bioactive agents for future tissue engineering applications.