Deepak M. Gupta

Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells

Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However, most of the studies used skin fibroblasts as the starting population for reprogramming, which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore, iPS cells can be readily derived from adult hASCs in a feeder-free condition, thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion, are easy to maintain in culture, and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells.

Cranial sutures: a brief review.

Summary: Craniosynostosis, or the premature fusion of one or more cranial sutures, is a relatively common congenital defect that causes a number of morphologic and functional abnormalities. With advances in genetics and molecular biology, research of craniosynostosis has progressed from describing gross abnormalities to understanding the molecular interactions that underlie these cranial deformities. Animal models have been extremely valuable in improving our comprehension of human craniofacial morphogenesis, primarily by human genetic linkage analysis and the development of knock-out animals. This article provides a brief review of perisutural tissue interactions, embryonic origins, signaling molecules and their receptors, and transcription factors in maintaining the delicate balance between proliferation and differentiation of cells within the suture complex that determines suture fate. Finally, this article discusses the potential implications for developing novel therapies for craniosynostosis.

Bone Regeneration and Repair

In the face of mounting clinical demand, and armed with reconstructive techniques that are technically challenging and frequently result in suboptimal patient outcomes, increasing focus is being placed on tissue engineering and regenerative medicine as a potential source of novel skeletal reconstructive approaches. Specifically, evidence is accumulating that highlights the promise of osteoprogenitor cell-based reconstructive strategies to meet the needs of an expanding patient population. Historically, the study of cell and molecular biology guiding physiologic and pathologic skeletal development, as well as endogenous bone regeneration following injury, has provided a wealth of information that lends insight toward potential parallel processes that may regulate the osteogenic differentiation of progenitor cells. Multiple progenitor cell populations are now known to possess a capacity to undergo robust osteogenic differentiation in the presence of appropriate environmental cues (hESC, BMSC, ASC, etc.) Recent investigations have put forth multiple advantages of ASC relative to BMSC. Of note, ASC exist in relative abundance, lack the need for in vitro expansion prior to utilization, and can be harvested with relative ease and reduced donor morbidity. Collectively, these factors, paired with promising in vitro and in vivo observations that speak toward the substantial osteogenic potential of ASC, have spurred enthusiasm to pursue the application of ASC in the maturation of skeletal tissue engineering applications. Yet, elucidating what structural and functional properties of scaffolds designed for ASC-mediated skeletal tissue engineering applications (porosity, pore size, composition, mechanical stability, degradation kinetics, etc.), as well as evolving our understanding and capacity to deliver spatiotemporally specific pro-osteogenic targeted molecular manipulation to progenitor cells, remain important hurdles to clear. The scope of this review encompasses the current state of ongoing investigations along these fronts, as well as what future direction will be critical to the transition of cell-based skeletal tissue engineering strategies to the bedside.

Tissue Engineering in Cleft Palate and Other Congenital Malformations

Contributions from multidisciplinary investigations have focused attention on the potential of tissue engineering to yield novel therapeutics. Congenital malformations, including cleft palate, craniosynostosis, and craniofacial skeletal hypoplasias represent excellent targets for the implementation of tissue engineering applications secondary to the technically challenging nature and inherent inadequacies of current reconstructive interventions. Apropos to the search for answers to these clinical conundrums, studies have focused on elucidating the molecular signals driving the biologic activity of the aforementioned maladies. These investigations have highlighted multiple signaling pathways, including Wnt, fibroblast growth factor, transforming growth factor-β, and bone morphogenetic proteins, that have been found to play critical roles in guided tissue development. Furthermore, a comprehensive knowledge of these pathways will be of utmost importance to the optimization of future cell-based tissue engineering strategies. The scope of this review encompasses a discussion of the molecular biology involved in the development of cleft palate and craniosynostosis. In addition, we include a discussion of craniofacial distraction osteogenesis and how its applied forces influence cell signaling to guide endogenous bone regeneration. Finally, this review discusses the future role of cell-based tissue engineering in the treatment of congenital malformations.

Applications of an athymic nude mouse model of nonhealing critical-sized calvarial defects.

Calvarial bone defects are a common clinical scenario in craniofacial surgery. Numerous approaches are used to reconstruct skull defects, and each possesses its own inherent disadvantages. This fact underscores the opportunity to develop a novel method to repair osseous defects in craniofacial surgery. Recent literature strongly suggests that cell-based therapies in the form of regenerative medicine may be a developing paradigm in reconstructive surgery. Although numerous studies have probed osteoprogenitor cells from mice, few have explored the biology of human cells in the setting of osteogenesis in an equally rigorous manner. This study proposes a nude mouse model of critical-sized calvarial defects to study the in vivo biology of human osteoprogenitor cells. Critical-sized 4.0-mm calvarial defects were created in nude mice (n = 15) with a custom trephine drill bit outfitted to a dental drill handpiece. During the craniotomy, the dura mater was spared from injury. Gross inspection, routine histology, and micro-computed tomographic scanning were performed at 2, 4, 8, and 16 weeks postoperatively. There was no calvarial healing in any of the animals by 16 weeks. The dura mater remained intact in all subjects. Gross, histologic, and radiographic assays confirmed these findings. Although several studies have implanted human osteoprogenitor cells in vivo in various animal models, few have documented the appropriate controls or conditions necessary to support the potential to translate benchtop findings into clinical applications. We propose in this study that the nude mouse critical-sized calvarial defect model will be valuable with increasing investigations with human osteoprogenitor cells.

Human Adipose-Derived Stromal Cells Respond to and Elaborate Bone Morphogenetic Protein-2 during In Vitro Osteogenic Differentiation

Background: Interest in the potential application of adipose-derived stromal cells in cell-mediated tissue engineering of bone and other mesenchymal-derived tissues is growing. This study aimed to investigate the hypothesis that human adipose-derived stromal cells respond to and elaborate bone morphogenetic protein (BMP) 2, which could represent an important target of molecular manipulation to enhance the osteogenic potential of human adipose-derived stromal cells. Methods: Human adipose-derived stromal cells were differentiated for 10 days toward the osteogenic lineage in osteogenic differentiation media alone or supplemented with recombinant human BMP2 (rhBMP2). Alizarin red staining was quantified by spectrophotometry. Gene expression analyses were performed using quantitative real-time polymerase chain reaction. BMP2 levels in conditioned media were titered by enzyme-linked immunosorbent assay daily during osteogenic differentiation. Human adipose-derived stromal cells were cultured in complete or partially (50 percent) changed osteogenic differentiation media, or unchanged osteogenic differentiation media, to assay for pro-osteogenic secreted factors. In addition, human adipose-derived stromal cells were cultured in osteogenic differentiation media supplemented with BMP2/BMP4-neutralizing antibody. Results: Exogenous rhBMP2 significantly augmented the in vitro osteogenic potential of human adipose-derived stromal cells in a dose-dependent fashion, and significantly increased transcript levels of RUNX2 and osteocalcin. BMP2, BMP4, BMPR1B, and SMAD1/5 expression was significantly increased during differentiation. Enzyme-linked immunosorbent assay demonstrated significantly increased BMP2 elaboration during differentiation. Culture in conditioned osteogenic differentiation media led to significantly increased matrix mineralization. Mineralization was significantly decreased when osteogenic differentiation media was supplemented with a BMP2/BMP4-neutralizing antibody. Conclusions: These data strongly support that BMP signaling is dynamic and important during normal in vitro osteogenic differentiation of human adipose-derived stromal cells. Thus, BMP2 may be used to enhance the osteogenic differentiation of human adipose-derived stromal cells for bone tissue engineering. Future studies will examine the effect of rhBMP2 on osteogenic differentiation of human adipose-derived stromal cells in vivo.

Tissue Harvest by Means of Suction-assisted or Third-generation Ultrasound-assisted Lipoaspiration Has No Effect on Osteogenic Potential of Human Adipose-derived Stromal Cells

Background: Human adipose-derived stromal cells readily undergo osteogenic differentiation in vitro and in vivo. Thus, interest in their potential role in skeletal tissue engineering continues to escalate. Very little is known regarding the effects that energy delivered by means of third-generation ultrasound-assisted lipoaspiration may have on the osteogenic potential of these cells. The authors investigated whether differences in adipose-derived stromal cell yield, and the in vitro proliferation and osteogenic potential of these cells obtained by suction-assisted lipoaspiration or third-generation ultrasound-assisted lipoaspiration, exist. Methods: Adipose-derived stromal cells were harvested from lipoaspiration specimens of patients undergoing elective suction-assisted lipoaspiration and third-generation ultrasound-assisted lipoaspiration. Harvested cells were seeded to evaluate proliferative capacity and in vitro osteogenic potential. Alkaline phosphatase and alizarin red staining were performed to evaluate early and terminal osteogenic differentiation, respectively. Quantitative real-time polymerase chain reaction analysis was used to examine osteogenic gene expression patterns of RUNX2/CFBA1 (early differentiation) and osteocalcin (late differentiation). Results: No significant differences in the proliferative capacity (n = 3), alkaline phosphatase staining (n = 3), or extracellular matrix mineralization (n = 3) of suction-assisted lipoaspiration– or third-generation ultrasound-assisted lipoaspiration–derived cells were appreciated. Transcript levels of markers of early and terminal osteogenic differentiation were not significantly different (n = 3). Conclusions: These findings suggest that exposure of adipose-derived stromal cells to ultrasound energy during tissue harvest by means of third-generation ultrasound-assisted lipoaspiration does not impart a negative consequence toward their proliferative capacity or osteogenic potential. Thus, the cells harvested using third-generation ultrasound-assisted lipoaspiration are comparable to those obtained by means of suction-assisted lipoaspiration for use in the study of osteogenic differentiation and skeletal tissue engineering.

Mesenchymal cells for skeletal tissue engineering.

Background: Skeletal defects represent a significant socioeconomic burden to the US healthcare system. Current options for reconstructing osseous deficits have shortcomings. Objective: To review the use of mesenchymal stem cells for skeletal tissue engineering. Methods: We focused on the application of mesenchymal cells in skeletal regeneration, optimization of this technique, tropic effects of multipotent mesenchymal cells, and future directions. Results/conclusion: A number of cell-based modalities have been investigated. We have been interested in the role of adipose-derived stromal cells in bone regeneration and understanding the mechanisms behind osteogenic differentiation of progenitor cells and acceleration of this process. Future clinical applications of multipotent mesenchymal cells will depend on better understanding of the molecular signaling involved in osteogenic differentiation and maintaining pluripotency.

The SNaP System: Biomechanical and Animal Model Testing of a Novel Ultraportable Negative-Pressure Wound Therapy System

BACKGROUND Negative-pressure wound therapy is traditionally achieved by attaching an electrically powered pump to a sealed wound bed and applying subatmospheric pressure by means of gauze or foam. The Smart Negative Pressure (SNaP) System (Spiracur, Inc., Sunnyvale, Calif.) is a novel ultraportable negative-pressure wound therapy system that does not require an electrically powered pump. METHODS Negative pressure produced by the SNaP System, and a powered pump, the wound vacuum-assisted closure advanced-therapy system (Kinetic Concepts, Inc., San Antonio, Texas), were compared in vitro using bench-top pressure sensor testing and microstrain and stress testing with pressure-sensitive film and micro-computed tomographic scan analysis. In addition, to test in vivo efficacy, 10 rats underwent miniaturized SNaP (mSNaP) device placement on open wounds. Subject rats were randomized to a system activation group (approximately -125 mmHg) or a control group (atmospheric pressure). Wound measurements and histologic data were collected for analysis. RESULTS Bench measurement revealed nearly identical negative-pressure delivery and mechanical strain deformation patterns between both systems. Wounds treated with the mSNaP System healed faster, with decreased wound size by postoperative day 7 (51 percent versus 12 percent reduction; p < 0.05) and had more rapid complete reepithelialization (21 days versus 32 days; p < 0.05). The mSNaP device also induced robust granulation tissue formation. CONCLUSIONS The SNaP System and an existing electrically powered negative-pressure wound therapy system have similar biomechanical properties and functional wound-healing benefits. The potential clinical efficacy of the SNaP device for the treatment of wounds is supported.

Global age-dependent differences in gene expression in response to calvarial injury.

Children less than 2 years of age are capable of healing large calvarial defects, whereas adults have been found to lack this endogenous ability. In this study, we used microarray analysis to compare genomewide expression patterns during active regeneration after injury with calvaria in skeletally immature and mature mice. Parietal bone defects were created in 6-day-old (juvenile) and 60-day-old (adult) mice using a 4-mm trephine bit (n = 20 mice per age group). The calvarial disc was removed, leaving the underlying dura mater intact. Two weeks after injury, the region of regeneration with the underlying dura mater was harvested, and RNA was extracted for microarray analysis. The 25 most differentially upregulated genes in juvenile regenerates compared with adults were listed, as well as selected bone-related genes. In addition, QRT-PCR confirmation of specific genes was performed for validation. Juvenile regenerates expressed significantly greater amounts of BMP-2, -4, -7, as well as FGF-2 and its receptor FGFR-1. Various other growth factors were also noted to be upregulated, including IGF-2 and Ptn. This corresponded with the increased expression of markers for osteogenic differentiation of Sparc and Oc. Markers of osteoclast activity, Acp5, Ctsk, and Mmp2, were noted to be greater in juvenile regenerates compared with adults. The observation of Mmp14 upregulation, however, highlights the importance of balanced osteoclast-mediated bone resorption for ultimate healing. The 2 most differentially regulated genes, transthyretin (Ttr) and prostaglandin D2 synthase (Ptgds), highlight the potential role of retinoic acid signaling and the prostaglandin axis on skeletal regeneration. These findings underscore the multitude of biomolecular mechanisms at play, allowing juvenile calvaria to heal after injury. The identification of various growth factors and cytokines involved also suggests novel therapeutic strategies for tissue-engineering purposes.