Peter Ashjian

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.

IN VITRO DIFFERENTIATION OF HUMAN PROCESSED LIPOASPIRATE CELLS INTO EARLY NEURAL PROGENITORS

Human processed lipoaspirate (PLA) cells are multipotent stem cells, capable of differentiating into multiple mesenchymal lineages (bone, cartilage, fat, and muscle). To date, differentiation to nonmesodermal fates has not been reported. This study demonstrates that PLA cells can be induced to differentiate into early neural progenitors, which are of an ectodermal origin. Undifferentiated cultures of human PLA cells expressed markers characteristic of neural cells such as neuron-specific enolase (NSE), vimentin, and neuron-specific nuclear protein (NeuN). After 2 weeks of treatment of PLA cells with isobutylmethylxanthine, indomethacin, and insulin, about 20 to 25 percent of the cells differentiated into cells with typical neural morphologic characteristics, accompanied by increased expression of NSE, vimentin, and the nerve-growth factor receptor trk-A. However, induced PLA cells did not express the mature neuronal marker, MAP, or the mature astrocyte marker, GFAP. It was also found that neurally induced PLA cells displayed a delayed-rectifier type K+ current (an early developmental ion channel) concomitantly with morphologic changes and increased expression of neural-specific markers. The authors concluded that human PLA cells might have the potential to differentiate in vitro into cells that represent early progenitors of neurons and/or glia.

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.

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.

The effect of postoperative anticoagulation on microvascular thrombosis.

Background:Numerous protocols exist to prevent thrombosis after free-tissue transfer. Many surgeons advocate using aspirin or other antiplatelet agents, but little objective evidence supports this practice. This study evaluates the rate of microvascular thrombosis in patients undergoing free-tissue transfer treated with or without antiplatelet agents. Methods:All consecutive free flaps from 2002–2005 at a single center were reviewed using a prospectively maintained database. Patients were in 2 groups based on postoperative anticoagulation administration. In group 1, 325 mg of aspirin was administered daily for 5 days postoperatively. In group 2, patients were treated with 5000 units of low-molecular-weight heparin (LMWH) per day until ambulating. Patient demographics, procedure type, diagnosis, adjuvant treatment, and procedure type were recorded. Outcome variables included microvascular thrombosis, partial or total flap loss, hematoma, bleeding, deep venous thrombosis (DVT), pulmonary embolism, and death. Results:Four hundred seventy patients underwent 505 microvascular free flaps to reconstruct oncologic defects. Two hundred sixty flaps (group A) received postoperative aspirin therapy; 245 flaps (group B) received LMWH therapy. Both groups were statistically similar in their composition. No statistically significant difference was noted between the 2 groups when comparing outcome variables including microvascular thrombosis, partial or total flap loss, hematoma, bleeding, DVT, pulmonary embolism, and death. Conclusions:Postoperative anticoagulation choice has no statistically significant effect on the incidence of free flap complications, including bleeding, thromboembolism, and flap loss. We conclude that aspirin or LMWH therapy demonstrates equivalent outcomes when used as single-agent postoperative anticoagulation in oncologic free flap reconstruction.

Is the use of intraoperative heparin safe

Background: Despite the reliability of microvascular free tissue transfer, flap loss remains a significant concern. To improve outcome, various pharmacologic agents have been used to prevent microvascular thrombosis. The authors review their experience with intraoperative heparin therapy, specifically addressing the risks of hematoma, pedicle thrombosis, and flap loss rate. Methods: Results from consecutive free flaps performed over a 3-year period were reviewed using a prospectively maintained database. Patients were divided into two groups: group A received a bolus of 3000 units of intraoperative heparin 10 minutes before flap pedicle ligation; group B did not receive intraoperative heparin. Patient demographics, procedure type, diagnosis, adjuvant treatment, and complications were recorded. Outcome variables included microvascular thrombosis, total and partial flap loss, hematoma, seroma, pulmonary embolism, and death. Results: Four hundred seventy patients underwent 505 microvascular free flaps for reconstruction of oncologic defects. Reconstructed areas included head and neck (n = 288), trunk and breast (n = 151), upper extremity (n = 12), and lower extremity (n = 19). Of these, 260 flaps (group A) received an intraoperative heparin bolus, and 245 flaps (group B) received no intraoperative heparin bolus. There were no statistically significant differences in major and minor complications between the two groups (power = 0.85). Conclusions: Intraoperative systemic heparin use has no statistically significant effect on the incidence of microvascular thrombosis. In addition, administration of a single dose of intraoperative heparin does not increase the rate of hematoma formation or prevent microvascular thrombosis. Thus, critical factors for flap survival are likely independent of the use of intraoperative anticoagulation.

Noninvasive in Situ Evaluation of Osteogenic Differentiation by Time-Resolved Laser-Induced Fluorescence Spectroscopy

The clinical implantation of bioengineered tissues requires an in situ nondestructive evaluation of the quality of tissue constructs developed in vitro before transplantation. Time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) is demonstrated here to noninvasively monitor the formation of osteogenic extracellular matrix (ECM) produced by putative stem cells (PLA cells) derived from human adipose tissue. We show that this optical spectroscopy technique can assess the relative expression of collagens (types I, III, IV, and V) within newly forming osteogenic ECM. The results are consistent with those obtained by conventional histochemical techniques (immunofluorescence and Western blot) and demonstrate that TR-LIFS is a potential tool for monitoring the expression of distinct collagen types and the formation of collagen cross-links in intact tissue constructs.

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.