Darko Bosnakovski

Isolation and multilineage differentiation of bovine bone marrow mesenchymal stem cells

The bone marrow harbors a population of mesenchymal stem cells (MSCs) that possess the potential to differentiate into bone, cartilage, and fat, and along other tissue pathways. To date, MSCs from various species have been studied. Despite the bovine experimental model being widely used in experiments in vivo and in vitro, only a limited amount of information regarding bovine MSCs is available. The aim of this study was to isolate and induce the multilineage mesenchymal differentiation of bovine MSCs, thereby initiating further research on these cells. Bovine MSCs were isolated from eight calves, and osteogenic, chondrogenic, and adipogenic differentiation was induced by using a combination of previously reported protocols for other species. The level of differentiation was evaluated by histological examination and by analyzing the expression of tissue-specific genes by a quantitative “real time” reverse transcription/polymerase chain reaction technique. Following osteoinduction, the isolated fibroblast-like cells transformed into cuboidal cells and formed alkaline-phosphatase-positive colonies; during differentiation, these colonies transformed into mineralized nodules. In addition, osteogenesis was followed by osteocalcin and collagen type I mRNA expression. Chondrogenesis was confirmed by the demonstration of collagen type II, aggrecan, and sox9 mRNA expression in the cells stimulated by transforming growth factor β1 in monolayer culture. After being cultured in an adipogenesis-inducing medium, the MSCs responded by the accumulation of lipid vacuoles and the expression of adipocyte-specific genes. We have therefore demonstrated that cells harvested from bovine bone marrow are capable of in vitro extensive multiplication and multilineage differentiation, making them a relevant and invaluable model in the field of stem cell research.

Inducible Cassette Exchange: A Rapid and Efficient System Enabling Conditional Gene Expression in Embryonic Stem and Primary Cells

Genetic modification is critically enabling for studies addressing specification and maintenance of cell fate; however, methods for engineering modifications are inefficient. We demonstrate a rapid and efficient recombination system in which an inducible, floxed cre allele replaces itself with an incoming transgene. We target this inducible cassette exchange (ICE) allele to the (HPRT) locus and demonstrate recombination in murine embryonic stem cells (ESCs) and primary cells from derivative ICE mice. Using lentivectors, we demonstrate recombination at a randomly integrated ICE locus in human ESCs. To illustrate the utility of this system, we insert the myogenic regulator, Myf5, into the ICE locus in each platform. This enables efficient directed differentiation of mouse and human ESCs into skeletal muscle and conditional myogenic transdetermination of primary cells cultured in vitro. This versatile tool is thus well suited to gain‐of‐function studies probing gene function in the specification and reprogramming of cell fate. STEM CELLS 2011;29:1580–1588

Relationship of Disease Progression and Plasma Histamine Concentrations in 11 Dogs with Mast Cell Tumors

Plasma histamine concentrations (PHCs) were measured serially over 9 months or until death in 11 dogs with mast cell tumors (MCTs). Eight dogs had grossly visible disease and the other 3 dogs had microscopic disease. Initial PHCs in the dogs with gross disease were significantly higher than PHCs in healthy dogs (median, 0.73 ng/mL and 0.19 ng/mL respectively; P < .009), whereas initial PHCs in dogs with microscopic disease showed no difference from controls. Seven dogs subsequently had progressive increases in PHC, and developed hyperhistaminemia (median, 14.0 ng/mL; range, 5.11-30.1 ng/nL). These 7 dogs died from MCTs, and 1 had general weakness with rapid lysis of a large tumor burden after radiation therapy. PHCs of the other 4 dogs were less than 1 ng/mL during the study. These 4 dogs were still alive with adequate control of the tumor at the conclusion of the study. Four of the 11 dogs initially had gastrointestinal (G1) signs, which abated soon after administration of histamine-2 (H-2) blockers. No significant difference was found between PHCs in dogs with GI signs and those without GI signs (median, 0.86 ng/mL and 0.35 ng/mL. respectively). Thereafter, 7 dogs had serious GI complications for which H-2 blocker therapy was ineffective. PHCs in these 7 dogs were extremely high (median, 12.2 ng/mL; range, 3.42-30.1 ng/nL). Results of this study demonsrated that PHC was one factor related to disease progression, and indicated that marked hyperhistaminemia was associated with the GI signs refractory to H-2 blocker therapy in dogs with MCTs.

Mesenchymal stem cells for anti-cancer drug delivery.

Self renewal, extensive proliferation and multilineage differentiation ability in vitro and in vivo make mesenchymal stem cells (MSCs) powerful tools for tissue engineering. Beyond their potential uses in regenerative medicine, an emerging field of research aims to utilize MSCs for anti-cancer treatment. These strategies are based on the remarkable ability of MSCs to localize and integrate into tumor stroma and deliver anti-cancer agents (US20100055167, US20120207725, US20120010499). Genetically engineered MSCs can specifically target different tumor types and locally secrete therapeutic proteins such as interferons α and β, interleukins 2 and 12 or chemokine CX3CL1 (US20110027239, US20120087901, WO2012071527). In addition, MSCs have also been engineered to deliver oncolytic viruses, for targeted chemotherapy using enzyme prodrug conversion or for inducing tumor cell apoptosis by delivering tumor necrosis factor-related apoptosis inducing ligand (TRAIL) (WO2012106281). The patent databases FPO and Delphion were used to locate patents that were published between 2005 and 2013. Here, we present the current progress and the most recent patents on MSC anti-cancer drug delivery systems and discuss future directions in the field.

Expression of the Human FSHD-Linked DUX4 Gene Induces Neurogenesis During Differentiation of Murine Embryonic Stem Cells

Misexpression of the double homeodomain protein DUX4 in muscle is believed to cause facioscapulohumeral muscular dystrophy (FSHD). Although strategies are being devised to inhibit DUX4 activity in FSHD, there is little known about the normal function of this protein. Expression of DUX4 has been reported in pluripotent cells and testis. To test the idea that DUX4 may be involved in initiating a germ lineage program in pluripotent cells, we interrogated the effect of expressing the human DUX4 gene at different stages during in vitro differentiation of murine embryonic stem (ES) cells. We find that expression of even low levels of DUX4 is incompatible with pluripotency: DUX4-expressing ES cells downregulate pluripotency markers and rapidly differentiate even in the presence of leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP4). Transcriptional profiling revealed unexpectedly that DUX4 induced a neurectodermal program. Embryoid bodies exposed to a pulse of DUX4 expression displayed severely inhibited mesodermal differentiation, but acquired neurogenic potential. In a serum-containing medium in which neurogenic differentiation is minimal, DUX4 expression served as a neural-inducing factor, enabling the differentiation of Tuj1+ neurites. These data suggest that besides effects in muscle and germ cells, the involvement of DUX4 in neurogenesis should be considered as anti-DUX4 therapies are developed.

Advancing with ceramic biocomposites for bone graft implants

Bone grafting has come a very long way since a Dutch surgeon used pieces of a dogs skull to repair a soldiers cranium in the 17 th Century. Current technology aims to deliver a scaffold that combines the unique osteogenic properties of ceramic biocomposite materials to make the best mimic of physiologic conditions. To do so, a scaffold must provide: i) A three-dimensional platform allowing for osteogenic cellular attachment and growth and vascular formation, ii) Struc- tural integrity while the damaged tissue heals, and iii) Non-toxic integration, degradation or resorption into the host over an appropriate time. The combination of inorganic, ceramic materials with cells, polymers and growth factors has come very close to creating a bone graft capable of meeting each of these requirements. Recent patents describe new methods to forming an ideal osteogenic matrix for both large and small bone repair. Many new technologies have been introduced that are very potent in their ability to heal small bone wounds and induce new bone formation, such as porous calcium phosphate pastes and hydroxyapatite cements. However, there is still a lack of quality and proven materials for load bear- ing purposes. This is a reminder of how much there still is to improve upon and that we are still a long way from creating bone products that are identical to the natural product. Despite these shortcomings, ceramic biocomposties represent one of the most promising materials in the bone graft field and their development and improvement will surely lead to a more natural bone replacement.

Author Correction: Muscle pathology from stochastic low level DUX4 expression in an FSHD mouse model

In the originally published version of this Article, an incorrect grant number, RO1 NS083549, was acknowledged. The correct grant number is RO1 AR055685. This error has now been corrected in both the PDF and HTML versions of the Article.

Myogenic differentiation of FSHD patient specific induced pluripotent stem cells

Human induced pluripotent stem (IPS) cells overcome several disadvantages of human embryonic stem cells, including host specificity and ethical issues. Patient-specific IPS cells can be generated from every donor by using different cell types, making them a suitable tool for autologous cell therapy and tissue engineering. IPS cells generated from patients with genetic disorders capture the disease genotype in the cell, making them a good model for studying the pathology of the disease, especially during development, and testing different therapeutic approaches. FSHD is one of the most common inherited myopathies, caused by a contraction within a subtelomeric array of D4Z4 repeats 4q35.2. It is characterized by uneven and progressive weakness and atrophy of facial, shoulder and upper arm muscles. The D4Z4 repeat contains an intronless double homeobox gene named DUX4 (double homeobox, chromosome 4) which was recently shown to be expressed specifically in FSHD myoblasts, but not in unaffected control cells. DUX4 expressed in high levels induced rapid cell death and in the low levels interferes with myogenesis by missregulating MoyD and Myf5. Myoblasts expressing DUX4 failed to fuse and form terminally differentiated myotubes. We generated IPS from myoblasts and fibroblast from seven FSHD patients and five controls using four reprogramming factors, Sox2, Klf4, Oct4 and c-Myc. IPS cells were characterized by analyzing expression of pluripotent markers and formation of teratomas. From IPS cells we generated mesenchymal stem cells using EB culturing system and mesoderm inducing growth factors. Mesenchymal stem cells express specific surface markers including CD73 and CD105, and under specific condition were able for adipogenic and osteogenic differentiation. To induce myogenesis IPS-MSC we transfected with Myod or Myf5. Stably transfected cells were able to expand as myoblasts in myogenic proliferation medium. Generated myoblasts express specific marker CD56 and under differentiation condition in growth factor deprived medium were able to fuse and terminally differentiate in myotubes. Myoblasts generated from IPS cells transplanted in MDX mice were able to engraft and restore dystrofine expression.T identification of skeletal progenitor cells in the degenerated intervertebral disc (IVD) raises the possibility that the IVD contains a population of stem cells capable of regenerating nucleus pulposus (NP) cells or annulus fibrosus (AF) cells. The characterization of IVD stem/progenitor cells (DSCs) proposes important clinical implications for the management of IVD diseases and injuries. However, it is still unknown about the origin of these cells, which is a tissue-specific or degenerationinduced population. We show that healthy Rhesus macaque IVD harbors stem-like cells, which are capable of generating colonyforming unit, differentiation and self-renewal, the common criteria of stem cells. We demonstrate that biglycan and decorin reduce the susceptibility of DSCs to hypoxia-induced apoptosis by promoting the expression of hypoxia inducible factors. Our findings suggest that normal IVD possess a progenitor population that may rely on unique niche comprised of small leucine-rich proteoglycans (SLRPs) for low oxygen tension survival. Since loss of SLRP in mice leads to disc degeneration, this implies disc degeneration may be associated with a deregulation of progenitor activities.