MSCs derived from iPSCs with a modified protocol are tumor-tropic but have much less potential to promote tumors than bone marrow MSCs.

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However, tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs, especially those in cancer patients. To circumvent these issues, we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable, but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition, invasion, stemness, and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFß, downstream protumor factors, and hyaluronan and its cofactor TSG6, which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for "off-the-shelf" therapies and bioengineering applications.

Theobromine Upregulates Osteogenesis by Human Mesenchymal Stem Cells In Vitro and Accelerates Bone Development in Rats.

Theobromine (THB) is one of the major xanthine-like alkaloids found in cacao plant and a variety of other foodstuffs such as tea leaves, guarana and cola nuts. Historically, THB and its derivatives have been utilized to treat cardiac and circulatory disorders, drug-induced nephrotoxicity, proteinuria and as an immune-modulator. Our previous work demonstrated that THB has the capacity to improve the formation of hydroxyl-apatite during tooth development, suggesting that it may also enhance skeletal development. With its excellent safety profile and resistance to pharmacokinetic elimination, we reasoned that it might be an excellent natural osteoanabolic supplement during pregnancy, lactation and early postnatal growth. To determine whether THB had an effect on human osteoprogenitors, we subjected primary human bone marrow mesenchymal stem cells (hMSCs) to osteogenic assays after exposure to THB in vitro and observed that THB exposure increased the rate of osteogenesis and mineralization by hMSCs. Moreover, THB exposure resulted in a list of upregulated mRNA transcripts that best matched an osteogenic tissue expression signature as compared to other tissue expression signatures archived in several databases. To determine whether oral administration of THB resulted in improved skeletal growth, we provided pregnant rats with chow supplemented with THB during pregnancy and lactation. After weaning, offspring received THB continuously until postnatal day 50 (approximately 10 mg kg-1 day-1). Administration of THB resulted in neonates with larger bones, and 50-day-old offspring accumulated greater body mass, longer and thicker femora and superior tibial trabecular parameters. The accelerated growth did not adversely affect the strength and resilience of the bones. These results indicate that THB increases the osteogenic potential of bone marrow osteoprogenitors, and dietary supplementation of a safe dose of THB to expectant mothers and during the postnatal period could accelerate skeletal development in their offspring.

Characterization of a pluripotent stem cell-derived matrix with powerful osteoregenerative capabilities.

Approximately 10% of fractures will not heal without intervention. Current treatments can be marginally effective, costly, and some have adverse effects. A safe and manufacturable mimic of anabolic bone is the primary goal of bone engineering, but achieving this is challenging. Mesenchymal stem cells (MSCs), are excellent candidates for engineering bone, but lack reproducibility due to donor source and culture methodology. The need for a bioactive attachment substrate also hinders progress. Herein, we describe a highly osteogenic MSC line generated from induced pluripotent stem cells that generates high yields of an osteogenic cell-matrix (ihOCM) in vitro. In mice, the intrinsic osteogenic activity of ihOCM surpasses bone morphogenic protein 2 (BMP2) driving healing of calvarial defects in 4 weeks by a mechanism mediated in part by collagen VI and XII. We propose that ihOCM may represent an effective replacement for autograft and BMP products used commonly in bone tissue engineering.

Rapid Osteogenic Enhancement of Stem Cells in Human Bone Marrow Using a Glycogen-Synthease-Kinase-3-Beta Inhibitor Improves Osteogenic Efficacy In Vitro and In Vivo.

Non-union defects of bone are a major problem in orthopedics, especially for patients with a low healing capacity. Fixation devices and osteoconductive materials are used to provide a stable environment for osteogenesis and an osteogenic component such as autologous human bone marrow (hBM) is then used, but robust bone formation is contingent on the healing capacity of the patients. A safe and rapid procedure for improvement of the osteoanabolic properties of hBM is, therefore, sought after in the field of orthopedics, especially if it can be performed within the temporal limitations of the surgical procedure, with minimal manipulation, and at point-of-care. One way to achieve this goal is to stimulate canonical Wingless (cWnt) signaling in bone marrow-resident human mesenchymal stem cells (hMSCs), the presumptive precursors of osteoblasts in bone marrow. Herein, we report that the effects of cWnt stimulation can be achieved by transient (1-2 hours) exposure of osteoprogenitors to the GSK3ß-inhibitor (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO) at a concentration of 800 nM. Very-rapid-exposure-to-BIO (VRE-BIO) on either hMSCs or whole hBM resulted in the long-term establishment of an osteogenic phenotype associated with accelerated alkaline phosphatase activity and enhanced transcription of the master regulator of osteogenesis, Runx2. When VRE-BIO treated hBM was tested in a rat spinal fusion model, VRE-BIO caused the formation of a denser, stiffer, fusion mass as compared with vehicle treated hBM. Collectively, these data indicate that the VRE-BIO procedure may represent a rapid, safe, and point-of-care strategy for the osteogenic enhancement of autologous hBM for use in clinical orthopedic procedures. Stem Cells Translational Medicine 2018;7:342-353.

How stem cell composition in bone marrow aspirate relates to clinical outcomes when used for cervical spine fusion.

Anterior cervical discectomy and fusion (ACDF) is performed to relieve pain caused by degenerative disk disease and nerve obstruction. As an alternative to bone graft, autologous concentrated bone marrow aspirate (CBMA) is used to achieve vertebral fusion with a satisfactory success rate. This has been attributed in part to bone marrow-resident mesenchymal stromal cells (MSCs) with the capacity to differentiate into osteoblasts and generate bone tissue. To date, there has been no study comparing cellular yields, MSC frequencies and their osteogenic potential with ACDF outcome. Patients (n = 24) received ACDF with CBMA and allograft bone matrix. Colony forming unit fibroblast (CFU-F) and CFU-osteoblasts (CFU-O) assays were performed on CBMA samples to enumerate MSCs (CFU-F) and osteogenic MSCs (CFU-O). CFUs were normalized to CBMA volume to define yield and also to mononuclear cells (MNC) to define frequency. After 1-year, fusion rates were good (86.7%) with pain and disability improved. There was a negative relationship between MNC and CFU-F measurements with age of patient and CFU-Os negatively correlated with age in females but not males. Tobacco use did not affect CBMA but was associated with poorer clinical outcome. Surprisingly, we found that while high-grade fusion was not associated with CFU-O, it correlated strongly (p<0.0067) with CBMA containing the lowest frequencies of CFU-F (3.0x10(-6)-5.83x10(-5) CFU-F/MNC). MNC levels alone were not responsible for the results. These observations suggest that osteogenesis by human bone marrow is controlled by homeostatic ratio of MSCs to other cellular bone marrow components rather than absolute level of osteogenic MSCs, and that a lower ratio of MSCs to other cellular components in marrow tends to predict effective osteogenesis during ACDF. The results presented herein challenge the current dogma surrounding the proposed mechanism of MSCs in bone healing.

Three-dimensional in vitro modeling of malignant bone disease recapitulates experimentally accessible mechanisms of osteoinhibition.

Malignant bone disease (MBD) occurs when tumors establish in bone, causing catastrophic tissue damage as a result of accelerated bone destruction and inhibition of repair. The resultant so-called osteolytic lesions (OL) take the form of tumor-filled cavities in bone that cause pain, fractures, and associated morbidity. Furthermore, the OL microenvironment can support survival of tumor cells and resistance to chemotherapy. Therefore, a deeper understanding of OL formation and MBD progression is imperative for the development of future therapeutic strategies. Herein, we describe a novel in vitro platform to study bone-tumor interactions based on three-dimensional co-culture of osteogenically enhanced human mesenchymal stem cells (OEhMSCs) in a rotating wall vessel bioreactor (RWV) while attached to micro-carrier beads coated with extracellular matrix (ECM) composed of factors found in anabolic bone tissue. Osteoinhibition was recapitulated in this model by co-culturing the OEhMSCs with a bone-tumor cell line (MOSJ-Dkk1) that secretes the canonical Wnt (cWnt) inhibitor Dkk-1, a tumor-borne osteoinhibitory factor widely associated with several forms of MBD, or intact tumor fragments from Dkk-1 positive patient-derived xenografts (PDX). Using the model, we observed that depending on the conditions of growth, tumor cells can biochemically inhibit osteogenesis by disrupting cWnt activity in OEhMSCs, while simultaneously co-engrafting with OEhMSCs, displacing them from the niche, perturbing their activity, and promoting cell death. In the absence of detectable co-engraftment with OEhMSCs, Dkk-1 positive PDX fragments had the capacity to enhance OEhMSC proliferation while inhibiting their osteogenic differentiation. The model described has the capacity to provide new and quantifiable insights into the multiple pathological mechanisms of MBD that are not readily measured using monolayer culture or animal models.

The effects of the Er:YAG laser on trabecular bone micro-architecture: Comparison with conventional dental drilling by micro-computed tomographic and histological techniques.

Background: The use of lasers has become increasingly common in the field of medicine and dentistry, and there is a growing need for a deeper understanding of the procedure and its effects on tissue. The aim of this study was to compare the erbium-doped yttrium aluminium garnet (Er:YAG) laser and conventional drilling techniques, by observing the effects on trabecular bone microarchitecture and the extent of thermal and mechanical damage. Methods: Ovine femoral heads were employed to mimic maxillofacial trabecular bone, and cylindrical osteotomies were generated to mimic implant bed preparation. Various laser parameters were tested, as well as a conventional dental drilling technique. The specimens were then subjected to micro-computed tomographic (µCT) histomorphometic analysis and histology. Results: Herein, we demonstrate that mCT measurements of trabecular porosity provide quantitative evidence that laser-mediated cutting preserves the trabecular architecture and reduces thermal and mechanical damage at the margins of the cut. We confirmed these observations with histological studies. In contrast with laser-mediated cutting, conventional drilling resulted in trabecular collapse, reduction of porosity at the margin of the cut and histological signs of thermal damage. Conclusions: This study has demonstrated, for the first time, that mCT and quantification of porosity at the margin of the cut provides a quantitative insight into damage caused by bone cutting techniques. We further show that with laser-mediated cutting, the marrow remains exposed to the margins of the cut, facilitating cellular infiltration and likely accelerating healing. However, with drilling, trabecular collapse and thermal damage is likely to delay healing by restricting the passage of cells to the site of injury and causing localized cell death.

An allograft generated from adult stem cells and their secreted products efficiently fuses vertebrae in immunocompromised athymic rats and inhibits local immune responses.

BACKGROUND CONTEXT: Spine pain and the disability associated with it are epidemic in the United States. According to the National Center for Health Statistics, more than 650,000 spinal fusion surgeries are performed annually in the United States, and yet there is a failure rate of 15%-40% when standard methods employing current commercial bone substitutes are used. Autologous bone graft is the gold standard in terms of fusion success, but the morbidity associated with the procedure and the limitations in the availability of sufficient material have limited its use in the majority of cases. A freely available and immunologically compatible bone mimetic with the properties of live tissue is likely to substantially improve the outcome of spine fusion procedures without the disadvantages of autologous bone graft. PURPOSE: This study aimed to compare a live human bone tissue analog with autologous bone grafting in an immunocompromised rat model of posterolateral fusion. DESIGN/SETTING: This is an in vitro and in vivo preclinical study of a novel human stem cell-derived construct for efficacy in posterolateral lumbar spine fusion. METHODS: Osteogenically enhanced human mesenchymal stem cells (OEhMSCs) were generated by exposure to conditions that activate the early stages of osteogenesis. Immunologic characteristics of OEhMSCs were evaluated in vitro. The secreted extracellular matrix from OEhMSCs was deposited on a clinical-grade gelatin sponge, resulting in bioconditioned gelatin sponge (BGS). Bioconditioned gelatin sponge was used alone, with live OEhMSCs (BGS+OEhMSCs), or with whole human bone marrow (BGS+hBM). Efficacy for spine fusion was determined by an institutionally approved animal model using 53 nude rats. RESULTS: Bioconditioned gelatin sponge with live OEhMSCs did not cause cytotoxicity when incubated with immunologically mismatched lymphocytes, and OEhMSCs inhibited lymphocyte expansion in mixed lymphocyte assays. Bioconditioned gelatin sponge with live OEhMSC and BGS+hBM constructs induced profound bone growth at fusion sites in vivo, with a comparable rate of fusion with syngeneic bone graft (negative [0 of 10], BGS alone [0 of 10], bone graft [7 of 10], BGS+OEhMSC [10 of 15], and BGS+hBM [8 of 8]). CONCLUSIONS: Collectively, these studies demonstrate that BGS+OEhMSC constructs possess low immunogenicity and drive vertebral fusion with efficiency matching syngeneic bone graft in rodents. We also demonstrate that BGS serves as a promising scaffold for spine fusion when combined with hBM.

A simple critical-sized femoral defect model in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue. Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Bone regeneration with osteogenically enhanced mesenchymal stem cells and their extracellular matrix proteins.

Although bone has remarkable regenerative capacity, about 10% of long bone fractures and 25% to 40% of vertebral fusion procedures fail to heal. In such instances, a scaffold is employed to bridge the lesion and accommodate osteoprogenitors. Although synthetic bone scaffolds mimic some of the characteristics of bone matrix, their effectiveness can vary because of biological incompatibility. Herein, we demonstrate that a composite prepared with osteogenically enhanced mesenchymal stem cells (OEhMSCs) and their extracellular matrix (ECM) has an unprecedented capacity for the repair of critical-sized defects of murine femora. Furthermore, OEhMSCs do not cause lymphocyte activation, and ECM/OEhMSC composites retain their in vivo efficacy after cryopreservation. Finally, we show that attachment to the ECM by OEhMSCs stimulates the production of osteogenic and angiogenic factors. These data demonstrate that composites of OEhMSCs and their ECM could be utilized in the place of autologous bone graft for complex orthopedic reconstructions.

Behavioral characterization of attractin, a water-borne peptide pheromone in the genus aplysia.

Pheromones play a significant role in coordinating reproductive activity in many animals, including opisthobranch molluscs of the genus APLYSIA: Although solitary during most of the year, these simultaneous hermaphrodites gather into breeding aggregations during the reproductive season. The aggregations contain both mating and egg-laying animals and are associated with masses of egg cordons. The egg cordons are a source of pheromones that attract other Aplysia to the area, reduce their latency to mating, and induce egg laying. One of these water-borne egg cordon pheromones ("attractin") has been characterized and shown to be attractive in T-maze assays. Attractin is the first water-borne peptide pheromone characterized in invertebrates. In the current studies, behavioral assays were used to better characterize the attraction, and to examine whether attractin can induce mating. Although the two activities could be related (i.e., attraction occurring because animals were looking for a partner), this was not tested. T-maze assays showed that attractin works as part of a bouquet of odors: the peptide is attractive only when Aplysia brasiliana is part of the stimulus. The animal does not need to be a conspecific, perhaps explaining why multiple species may be associated with one aggregation. Native and recombinant attractin are equally attractive, verifying that N-glycosylation at residue 8 is not required for attraction. Mating studies showed that both native and recombinant attractin reduce the latency to mating. The effects are larger when hermaphroditic mating is considered: in addition to reducing latency, attractin doubles the number of pairs mating as hermaphrodites. The effect may result from attractin stimulating both animals to mate as males and would be consistent with behaviors previously seen in the T-maze. Attractin may thus be contributing to the formation of copulatory chains and rings seen in aggregations in the field. These results may be interpreted in two ways: (1). attractin has multiple activities that contribute to the establishment and maintenance of the aggregation; or (2). the induced desire to mate may make attractin attractive when it is presented in conjunction with an animal. In either case, the results open the door for cellular and molecular studies of mechanism of action.

Human mesenchymal stem cell-derived matrices for enhanced osteoregeneration.

The methodology for the repair of critical-sized or non-union bone lesions has unpredictable efficacy due in part to our incomplete knowledge of bone repair and the biocompatibility of bone substitutes. Although human mesenchymal stem cells (hMSCs) differentiate into osteoblasts, which promote bone growth, their ability to repair bone in vivo has been variable. We hypothesized that given the multistage process of osteogenesis, hMSC-mediated repair might be maximal at a specific time point of healing. Using a mouse model of calvarial healing, we demonstrate that the osteo-repair capacity of hMSCs can be substantially augmented by treatment with an inhibitor of peroxisome proliferator-activated receptor γ, but efficacy is confined to the rapid osteogenic phase. Upon entry into the bone-remodeling phase, hMSC retention signals are lost, resulting in truncation of healing. To solve this limitation, we prepared a scaffold consisting of hMSC-derived extracellular matrix (ECM) containing the necessary biomolecules for extended site-specific hMSC retention. When inhibitor-treated hMSCs were coadministered with ECM, they remained at the injury, well into the remodeling phase of healing, which resulted in reproducible and complete repair of critical-sized bone defects in mice in 3 weeks. These data suggest that hMSC-derived ECM and inhibitor-treated hMSCs could be used at optimal times to substantially and reproducibly improve bone repair.