Daniel A. De Ugarte

Comparison of Multi-Lineage Cells from Human Adipose Tissue and Bone Marrow

Our laboratory has recently characterized a population of cells from adipose tissue, termed processed lipoaspirate (PLA) cells, which have multi-lineage potential similar to bone-marrow-derived mesenchymal stem cells (MSCs). This study is the first comparison of PLA cells and MSCs isolated from the same patient. No significant differences were observed for yield of adherent stromal cells, growth kinetics, cell senescence, multi-lineage differentiation capacity, and gene transduction efficiency. Adipose tissue is an abundant and easily procured source of PLA cells, which have a potential like MSCs for use in tissue-engineering applications and as gene delivery vehicles.

Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow

Our laboratory has characterized a population of stromal cells obtained from adipose tissue termed processed lipoaspirate cells (PLAs). PLAs, like bone-marrow derived mesenchymal stem cells (BM-MSCs), have the capacity to differentiate along the adipogenic, osteogenic, chondrogenic, and myogenic lineages, In order to better characterize these two multi-lineage populations, we examined the surface phenotype of both bone marrow and adipose tissue-derived cells from five patients undergoing surgery. PLA and BM-MSC cells were isolated, subcultivated, and evaluated for cell surface marker expression using flow cytometry. PLA and BM-MSC cells both expressed CD13, CD29, CD44, CD90, CD105, SH-3, and STRO-1. Differences in expression were noted for cell adhesion molecules CD49d (Integrin alpha4), CD54 (ICAM-1), CD34, and CD106 (VCAM-1). While markedly similar, the surface phenotypes of PLA and BM-MSC cells are distinct for several cell adhesion molecules implicated in hematopoietic stem cell homing, mobilization, and proliferation.

Multilineage Cells from Adipose Tissue as Gene Delivery Vehicles

We have characterized a population of mesenchymal progenitor cells from adipose tissue, termed processed lipoaspirate (PLA) cells, which have multilineage potential similar to bone marrow-derived mesenchymal stem cells and are also easily expanded in culture. The primary benefit of using adipose tissue as a source of multilineage progenitor cells is its relative abundance and ease of procurement. We examined the infection of PLA cells with adenoviral, oncoretroviral, and lentiviral vectors. We demonstrate that PLA cells can be transduced with lentiviral vectors at high efficiency. PLA cells maintain transgene expression after differentiation into adipogenic and osteogenic lineages after lentiviral transduction. Therefore, PLA cells and lentiviral vectors may be an efficient combination for use as a therapeutic gene delivery vehicle.

In Vivo Distribution of Human Adipose-Derived Mesenchymal Stem Cells in Novel Xenotransplantation Models

The potential for human adipose‐derived mesenchymal stem cells (AMSC) to traffic into various tissue compartments was examined using three murine xenotransplantation models: nonobese diabetic/severe combined immunodeficient (NOD/SCID), nude/NOD/SCID, and NOD/SCID/MPSVII mice. Enhanced green fluorescent protein was introduced into purified AMSC via retroviral vectors to assist in identification of cells after transplantation. Transduced cells were administered to sublethally irradiated immune‐deficient mice through i.v., intraperitoneal, or subcutaneous injection. Up to 75 days after transplantation, tissues were harvested and DNA polymerase chain reaction (PCR) was performed for specific vector sequences as well as for human Alu repeat sequences. Duplex quantitative PCR using human β‐globin and murine rapsyn primers assessed the contribution of human cells to each tissue. The use of the novel NOD/SCID/MPSVII mouse as a recipient allowed rapid identification of human cells in the murine tissues, using an enzyme reaction that was independent of surface protein expression or transduction with an exogenous transgene. For up to 75 days after transplantation, donor‐derived cells were observed in multiple tissues, consistently across the various administration routes and independent of transduction parameters. Tissue localization studies showed that the primary MSC did not proliferate extensively at the sites of lodgement. We conclude that human AMSC represent a population of stem cells with a ubiquitous pattern of tissue distribution after administration. AMSC are easily obtained and highly amenable to current transduction protocols for retroviral transduction, making them an excellent avenue for cell‐based therapies that involve a wide range of end tissue targets.

Future of fat as raw material for tissue regeneration.

Tissue replacement traditionally requires use of autologous tissue and is associated with the attendant morbidity of donor site harvest. In the case of allograft transplantation, there are concerns, similar to those associated with organ transplantation, of rejection and immunosuppression. For these reasons, emphasis has been placed on the development of tissue-engineered substitutes that incorporate autologous stem cells into tissue-engineered scaffolds. The authors’ laboratory has characterized a population of cells obtained from processed lipoaspirate (PLA), which have the capacity in vitro to differentiate into osteoblasts, chondrocytes, myocytes, adipocytes, and neuron-like cells. 1 Adipose tissue is an abundant, expendable, and easily obtained tissue that may prove to be an ideal source of autologous stem cells for engineering tissues.

Robotic surgery and resident training

BACKGROUND Robotic technology promises to have an important future in surgery, but few residency programs incorporate robotics into surgical training. We sought to compare the speed and accuracy with which junior residents could perform laparoscopic tasks using both a robotic surgical device (Zeus MicroWrist) and conventional laparoscopic instruments. METHODS Twelve residents performed exercises of progressive difficulty in an inanimate model using both the robot and conventional laparoscopy. Analysis of variance statistical analysis was used to compare task time and suturing accuracy scores. RESULTS Grasping and suturing exercises were performed significantly faster with conventional laparoscopic instruments than with the robot. However, no difference in task time was noted for intracorporeal knot tying. Accuracy scores for suturing were higher for the robot. CONCLUSIONS Junior residents can be instructed easily and quickly in both robotic and conventional advanced laparoscopic skills. The utility of robotic surgical devices in resident training requires further investigation.

Surgisis patch tracheoplasty in a rodent model for tracheal stenosis.

BACKGROUND Tracheal stenosis is a challenging surgical problem that can require reconstruction using autologous grafts or artificial stents. In this study, we evaluate the efficacy of Surgisis, a commercially available, biocompatible, acellular matrix, in the repair of a critical-size tracheal defect. METHODS A full-thickness defect (2 mm x 6 mm) was created in tracheal rings 4 through 6 in adult rats. A piece of 8-ply Surgisis (Cook; Bloomington, IN) was sutured to the edges of the defect with interrupted 8-0 polypropylene sutures. In control animals, the defect was closed primarily. The trachea was harvested at 4 weeks and prepared for histologic evaluation using conventional techniques. Cross-sectional area and perimeters were calculated using imaging software. RESULTS Tracheal defects without patch repair (n = 3) resulted in tracheal stenosis and immediate death. Animals that underwent Surgisis patch repair of tracheal defects (n = 10) tolerated the procedure well and had no audible stridor or evidence of respiratory distress. Eight of ten animals survived 4 weeks. The tracheal lumen was patent with no evidence of contracture or degradation of the Surgisis. Histologically, neovascularization of the Surgisis was noted with moderate inflammation. The surface of the Surgisis patch was covered ith a lining of ciliated epithelial cells. CONCLUSION In the rodent model, Surgisis appears to be an efficacious method for the patch repair of partial circumferential tracheal defects. Surgisis appears to be a safe and promising means of facilitating neovascularization and tissue regeneration. The long-term use of Surgisis warrants further investigation.

Treatment of chronic wounds by local delivery of granulocyte-macrophage colony-stimulating factor in patients with neutrophil dysfunction

Abstract.Chronic wounds are associated with considerable morbidity and prolonged hospitalizations. The availability of recombinant growth factors and cytokines provides a new modality for treatment of recalcitrant wounds. Granulocyte-macrophage colony-stimulating factor (GM-CSF), a growth protein for hematopietic cells, also enhances neutrophil and monocyte function and promotes keratinocyte proliferation. In three patients with inherited disorders associated with leukocyte dysfunction and non-healing wounds, topical application of GM-CSF resulted in complete wound closure within 1 to 4 weeks. A subcutaneous (s.c.) infusion pump for the local s.c. delivery of GM-CSF was also found to enhance healing. Local application of GM-CSF may thus promote wound closure in patients with impaired wound healing.

Lipoplasty: From Body Contouring to Tissue Engineering

LEARNING OBJECTIVES The reader is presumed to have a broad understanding of plastic surgical procedures and concepts. After studying this article, the participant should be able to: Physicians may earn 1 hour of Category 1 CME credit by successfully completing the examination based on material covered in this article. The examination begins on page ***. BACKGROUND The rapid development of disciplines such as cell therapy and tissue engineering has focused attention on stem cells as the ideal cellular substrate for new tissues. Human adipose tissue is a potential source of such stem cells. OBJECTIVE We review the role of human adipose tissue in stem cell research and describe the procurement of stem cells from the stromal vascular fraction of human adipose tissue obtained through suction-assisted lipoplasty. METHODS Raw lipoaspirate obtained through suction-assisted lipoplasty was washed in phosphate-buffered saline and digested with collagenase. The collagenase was then inactivated by fetal bovine serum and the cells were centrifuged for 10 minutes at 1200 x g. The resulting cell pellet was resuspended, plated, and maintained in nondifferentiating control media. RESULTS Processing of 250 to 500 mL of suctioned tissue routinely yielded 2 to 6 x 10(8) processed lipoaspirate cells. Cell viability was typically >95%. These cells have been shown to differentiate in vitro into at least the adipogenic, chondrogenic, myogenic, neurogenic, and osteogenic lineages in the presence of specific induction factors. CONCLUSIONS Adipose tissue may be an ideal source of stem cells, because it is abundant, easy to obtain in large quantities, and safe to procure. Such a development could place the plastic surgeon at the epicenter of medical research. Issues that require further research include elucidation of site-specific differences in fat cells, the use of vacuum-assisted lipoplasty and ultrasound-assisted lipoplasty in procuring stem cells, and the development of more efficient and convenient tissue processing techniques. (Aesthetic Surg J 2002;22:121-127.).

Lifetime fluorescence spectroscopy for in situ investigation of osteogenic differentiation

Time-Resolved Laser-Induced Fluorescence Spectroscopy (TR-LIFS) represents a potential tool for the in-situ characterization of bioengineered tissues. In this study, we evaluate the application of TR-LIFS to non-intrusive monitoring of matrix composition during osteogenetic differentiation. Human adipose-derived stem cells, harvested from 3 patients, were induced in osteogenic media for 3, 5, and 7 weeks. Samples were subsequently collected and probed for time-resolved fluorescence emission with a pulsed nitrogen laser. Fluorescence parameters, derived from both spectral- and time-domain, were used for sample characterization. The samples were further analyzed using Western blot analysis and computer-based densitometry. A significant change in the fluorescence parameters was detected for samples beyond 3 weeks of osteogenic differentiation. The spectroscopic observations: 1) show increase of collagen I when contrasted against the time-resolved fluorescence spectra of commercially available collagens; and 2) are in agreement with Western blot analysis that demonstrated significant increase in collagen I content between 3- vs. 5-weeks and 3- vs. 7-weeks and no changes for collagens III, IV, and V. Our results suggest that TR-LIFS can be used as a non-invasive means for the detection of specific collagens in maturing connective tissues.