MAPK signaling has stage-dependent osteogenic effects on human adipose-derived stem cells in vitro.

OVERVIEW: The use of pro-osteogenic growth factors, such as BMP2, in human adipose-derived stem cell (ASC) osteogenesis is well described. Because these growth factors work via signal transduction pathways, such as the mitogen-activated protein kinase (MAPK) cascade, a study of the relationship between MAPK signaling and ASC osteogenesis was conducted. MATERIALS AND METHODS: ERK, JNK, and p38MAPK activation were measured in ASCs osteo-induced using either dexamethasone or vitamin D3 and correlated with mineralization. Activation and mineralization were also measured without dexamethasone or using the glucocorticoid, cortisone. The expression of the MAPK phosphatase, MKP1, and its relationship to mineralization was also assessed. The effect of decreasing MAPK activation on mineralization through the use of exogenous inhibitors was examined along with siRNA-knockdown and adenoviral overexpression of ERK1/2. Finally, the effect of ERK1/2 overexpression on ASCs induced on PLGA scaffolds was assessed. RESULTS: ASC mineralization in dexamethasone or vitamin D3-induced ASCs correlated with both increased ERK1/2 and JNK1/2 activation. ASCs induced without dexamethasone also mineralized, with JNK1/2 signaling possibly mediating this event. No link between cortisone induction and MAPK signaling could be ascertained. ASCs treated with ERK, JNK, or p38MAPK inhibitors showed decreased osteogenic gene expression and diminished mineralization. Mineralization levels were also affected by viruses designed to inhibit or augment ERK1/2 expression and activity. Finally, ASC mineralization appeared to be a balance between the MAPK kinase activity and MKP1. CONCLUSIONS: It is likely that MAPK signaling plays a significant role in ASC osteogenesis, affecting differentiation in kinase- and stage-specific manners.

Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment.

Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion.

Adipose-derived stem cells and BMP2: part 1. BMP2-treated adipose-derived stem cells do not improve repair of segmental femoral defects.

Recombinant human bone morphogenetic protein-2 (rhBMP2) has been shown to induce both in vitro osteogenic differentiation and in vivo bone formation, with the capacity of rhBMP2 to elicit the repair of numerous bony defects (calvaria, spinal fusion, femora, and so on) well documented. In addition, rhBMP2 has been approved by the Food and Drug Administration (FDA) for selected human indications. Despite the fact that healing is often achieved, the challenge still remains to optimize the therapeutic use of rhBMP2. One avenue may be through the combination of rhBMP2 with stem cells capable of osteogenic differentiation. This study investigates the ability of rhBMP2 at various doses in combination with human adipose-derived stem cells (ASCs) to heal critical-sized rat segmental femoral defects. For this, different doses of rhBMP2 were incorporated with apatite-coated porous poly(l-lactide-co-dl-lactide) (70 : 30) (PLDLA) scaffolds, seeded with ASCs, and implanted into athymic rats. After 8 weeks, all implants were harvested and processed for bone formation using micro computed tomography (microCT) analysis and histology. Despite the findings that indicate no adverse effect of the apatite surface on ASC osteogenesis, no significant difference in bone formation could be qualitatively or quantitatively determined upon the implantation of ASC-seeded scaffolds absorbed to increasing doses of rhBMP2. Such results would suggest that the presence of ASCs within rhBMP2-absorbed scaffolds does not improve the bone-forming ability of the construct and that the formation of bone may be driven by the rhBMP2 alone. Based on these results, the addition of ASCs to rhBMP2-treated scaffolds may provide no significant advantage in terms of the ability to heal bone.

Adipose-derived stem cells and BMP2: part 2. BMP2 may not influence the osteogenic fate of human adipose-derived stem cells.

Although recent studies have proposed that human adipose-derived stem cells (ASCs), together with BMP2, can heal critical-sized bony defects, a companion study in this issue suggests that ASCs may not respond to BMP2 in vivo. To examine why this may be occurring. ASCs were treated with BMP2 and the cells' in vitro osteogenic capacity assessed along with the canonical BMP2 signaling pathway. In vitro treatment of ASCs with BMP2 had no consistent and significant effect on matrix mineralization or their expression of several osteogenic markers. Consistent and significant changes to Smad1/5/8 phosphorylation levels were also not observed upon BMP2 induction. The removal of dexamethasone from the BMP2 induction conditions had no effect on the observed results nor did stimulating ASCs with BMP2 from an alternate source (INFUSE; Medtronic, Minneapolis, MN, USA). In addition, no BMP-induced nuclear translocation of Smad1/Smad4 complexes could be discerned, suggesting that the canonical BMP2 signaling pathway may not be functional in ASCs. Interestingly, three downstream BMP2 pathway genes, distal-less3 (dlx3), distal-less5 (dlx5), and osterix, were not expressed in BMP2-induced ASCs, calling the utility of BMP2 induced in ASCs into question. The results of this in vitro study were consistent with that of our companion in vivo study that suggests a lack of effect of BMP2 on the osteogenic capacity of ASCs. Taken together, the data from both studies suggest that ASC osteogenic differentiation may not be influenced by BMP2. Consequently, combining BMP2 treatment with adult stem cells, like ASCs, may not be a viable strategy for bony healing.

Differential effect of ionizing radiation exposure on multipotent and differentiation-restricted bone marrow mesenchymal stem cells.

Debilitating effects of bone marrow from ionizing radiation exposure has been well established for hematopoietic stem cells; however, radiation toxicity of mesenchymal stem cells (MSCs) has been controversial. The present study addressed if ionizing radiation exposure differently affected bone marrow MSCs with various differentiation commitments. Mouse bone-marrow-derived MSCs, D1 cells of early passages (≤ 5 passages; p5) maintained the complete characteristics of multipotent MSCs, whereas, after ≥ 45 passages (p45) the differentiation capability of D1 cells became partially restricted. Both p5 and p45 D1 cells were subjected to single dose irradiation by radioactive isotope (137)Cs. Radiation treatment impaired cell renewal and differentiation activities of p5 D1 cells; however, p45 D1 cells were less affected. Radiation treatment upregulated both pro- and anti-apoptotic genes of p5 D1 cells in a dose-dependent manner, potentially resulting in the various apoptosis thresholds. It was found that constitutive as well as radiation-induced phosphorylation levels of histone H2AX was significantly higher in p45 D1 cells than in p5 D1 cells. The increased repair activity of DNA double-strand breakage may play a role for p45 D1 cells to exhibit the relative radioresistance. In conclusion, the radiation toxicity predominantly affecting multipotent MSCs may occur at unexpectedly low doses, which may, in part, contribute to the catabolic pathology of bone tissue.

Human levator veli palatini muscle: a novel source of mesenchymal stromal cells for use in the rehabilitation of patients with congenital craniofacial malformations.

BACKGROUND: Bone reconstruction in congenital craniofacial differences, which affect about 2-3% of newborns, has long been the focus of intensive research in the field of bone tissue engineering. The possibility of using mesenchymal stromal cells in regenerative medicine protocols has opened a new field of investigation aimed at finding optimal sources of multipotent cells that can be isolated via non-invasive procedures. In this study, we analyzed whether levator veli palatini muscle fragments, which can be readily obtained in non-invasive manner during palatoplasty in cleft palate patients, represent a novel source of MSCs with osteogenic potential. METHODS: We obtained levator veli palatini muscle fragments (3-5 mm3), during surgical repair of cleft palate in 5 unrelated patients. Mesenchymal stromal cells were isolated from the muscle using a pre-plating technique and other standard practices. The multipotent nature of the isolated stromal cells was demonstrated via flow cytometry analysis and by induction along osteogenic, adipogenic, and chondrogenic differentiation pathways. To demonstrate the osteogenic potential of these cells in vivo, they were used to reconstruct a critical-sized full-thickness calvarial defect model in immunocompetent rats. RESULTS: Flow cytometry analysis showed that the isolated stromal cells were positive for mesenchymal stem cell antigens (CD29, CD44, CD73, CD90, and CD105) and negative for hematopoietic (CD34 and CD45) or endothelial cell markers (CD31). The cells successfully underwent osteogenic, chondrogenic, and adipogenic cell differentiation under appropriate cell culture conditions. Calvarial defects treated with CellCeram™ scaffolds seeded with the isolated levator veli palatini muscle cells showed greater bone healing compared to defects treated with acellular scaffolds. CONCLUSION: Cells derived from levator veli palatini muscle have phenotypic characteristics similar to other mesenchymal stromal cells, both in vitro and in vivo. Our findings suggest that these cells may have clinical relevance in the surgical rehabilitation of patients with cleft palate and other craniofacial anomalies characterized by significant bone deficit.

The intracellular distribution of the ES cell totipotent markers OCT4 and Sox2 in adult stem cells differs dramatically according to commercial antibody used.

To characterize ES cells, researchers have at their disposal a list of pluripotent markers, such as OCT4. In their quest to determine if adult stem cell populations, such as MSCs and ASCs, are pluripotent, several groups have begun to report the expression of these markers in these cells. Consistent with this, human ASCs (hASCs) are shown in this study to express a plethora of ES pluripotent markers at the gene and protein level, including OCT4, Sox2, and Nanog. When intracellular distribution is examined in hASCs, both OCT4 and Sox2 are expressed within the nuclei of hASCs, consistent with their expression patterns in ES cells. However, a significant amount of expression can be noted within the hASC cytoplasm and a complete absence of nuclear expression is observed for Nanog. Recent descriptions of OCT4 transcript variants may explain the cytoplasmic expression of OCT4 in hASCs and consistent with this, hASCs do express both the OCT4A and 4B transcript variants at the gene level. However, discrepancies arise when these three pluripotent markers are studied at the protein level. Specifically, distinct differences in intracellular expression patterns were noted for OCT4, Sox2, and Nanog from commercial antibody to commercial antibody. These antibody discrepancies persisted when hMSCs and rat ASCs and MSCs were examined. Therefore, confirming the expression of OCT4, Sox2, and Nanog in adult stem cells with today's commercial antibodies must be carefully considered before the designation of pluripotent can be granted.

Freezing adipose tissue grafts may damage their ability to integrate into the host.

In this study, the effect of freezing on the morphology, viability, and VEGF synthesis of human adipose tissue grafts is examined. Currently, storage of adipose grafts involves freezing in simple saline solutions. However, the effect of freezing on the morphology and function of adipose tissue remains unclear. As a result, this study attempts to determine whether freezing adipose grafts should be considered prior to soft-tissue augmentation. In this study, the freezing of adipose grafts in saline for only 24 hr resulted in morphological changes in vivo and affected their ability to synthesize VEGF. The use of a simple cryopreservation medium containing sucrose appeared to maintain VEGF synthetic levels by the grafts and improved both their morphology and retention in vivo. However, the benefits of this cryopreservation medium were directly linked to storage time as long-term storage did not result in any noticeable benefit to graft retention. Finally, as an alternative to freezing, adipose grafts were combined with human adipose-derived stem cells (ASCs) to determine if their presence could enhance in vivo graft structure. The presence of ASCs did appear to improve graft structure in vivo over the short term and was also capable of improving tissue morphology when combined with grafts frozen in PBS. In conclusion, the successful use of adipose grafts may require a closer examination of the graft's storage conditions and time. Specifically, it now appears that the practice of freezing in saline may not be advisable if graft viability, activity, and structure are to be maintained in vivo.

Human adipose stem cells: a potential cell source for cardiovascular tissue engineering.

BACKGROUND/AIMS: A crucial step in providing clinically relevant applications of cardiovascular tissue engineering involves the identification of a suitable cell source. The objective of this study was to identify the exogenous and endogenous parameters that are critical for the differentiation of human adipose stem cells (hASCs) into cardiovascular cells. METHODS: hASCs were isolated from human lipoaspirate samples, analyzed, and subjected to two differentiation protocols. RESULTS: As shown by fluorescence-activated cell sorter (FACS) analysis, a population of hASCs expressed stem cell markers including CXCR4, CD34, c-kit, and ABCG2. Further, FACS and immunofluorescence analysis of hASCs, cultured for 2 weeks in DMEM-20%-FBS, showed the expression of smooth muscle cell (SMC)-specific markers including SM alpha-actin, basic calponin, h-caldesmon and SM myosin. hASCs, cultured for 2 weeks in endothelial cell growth medium-2 (EGM-2), formed a network of branched tube-like structures positive for CD31, CD144, and von Willebrand factor. The frequency of endothelial cell (EC) marker-expressing cells was passage number-dependent. Moreover, hASCs attached and formed a confluent layer on top of electrospun collagen-elastin scaffolds. Scanning electron microscopy and DAPI staining confirmed the integration of hASCs with the fibers and formation of a cell-matrix network. CONCLUSION: Our results indicate that hASCs are a potential cell source for cardiovascular tissue engineering; however, the differentiation capacity of hASCs into SMCs and ECs is passage number- and culture condition-dependent.

The role of cytoprotective cytokines in cardiac ischemia/reperfusion injury.

The mechanism(s) underlying the beneficial effects of adult mesenchymal stem cells (MSCs) after myocardial infarction (MI) is poorly understood. One possible explanation is the ability of MSCs to secrete cytokines, which modulate cardiomyocyte survival and function. MSCs express at least two cytoprotective cytokines, hepatocyte growth factor (HGF) and stromal cell-derived factor-1 alpha (CXCL12). The aim of our study was to compare the effects of these two cytokines administered acutely post-MI. We subjected adult male Lewis rats to myocardial ischemia/reperfusion injury. Immediately upon reperfusion, polymers saturated with HGF or CXCL12 were placed onto the infarcted anterior wall and the rats were allowed to recover. Echocardiographic analysis at 4 wk post-MI to assess left ventricular (LV) function revealed that LV ejection fraction was increased in the HGF treated group compared with the phosphate-buffered saline (PBS) control group. Likewise, LV end diastolic dimension was reduced in the HGF treated group compared with the PBS control group. Similarly, invasive hemodynamics at 12 wk showed improved contractility and relaxation in the HGF treated group compared with the PBS control group. In contrast, no significant effect on LV function was seen in the CXCL12 treated group. To determine the potential mechanism for this effect, infarct size (IFS) at 72 h was determined. IFS was decreased 4.2-fold in the HGF treated group compared with the PBS control group. Thus, HGF acutely post-MI using polymer delivery reduces IFS, leading to beneficial effects on post-MI LV remodeling.

Healing full-thickness cartilage defects using adipose-derived stem cells.

The purpose of this study was to evaluate the use of adipose-derived stem cells (ADSCs) as a source for full-thickness cartilage repair in an animal model. Autologous ADSCs were isolated and induced with growth medium and placed in a fibrin glue scaffold and into 3-mm x 4-mm full-thickness chondral defects in rabbits with negative controls. Specimens were evaluated for early healing using immunostaining, Western blotting, reverse transcriptase polymerase chain reaction, transfection with the Lac Z gene, and quantitative assessment. Twelve of 12 (100%) articular surface defects containing tissue-engineered stem cell constructs healed with hyaline-like cartilage, versus 1 of 12 (8%) in the control group (p < .001). There was complete healing to subchondral bone in 12 of 12 experimental defects (100%), and 10 of 12 (83%) had seamless annealing to the native cartilage. Aggrecan, superficial zone protein, collagen type II messenger ribonucleic acid, and Lac-Z gene products were identified in 12 of 12 experimental specimens, which exhibited a collagen type II:I protein ratio similar to that of normal rabbit cartilage. Quantitative histologic analysis revealed an average score of 18.2 of 21 in the experimental group, compared with 10.0 in the controls (p = .001). Induced ADSCs supported in a fibrin glue matrix are a promising cell source for cartilage tissue engineering.

Downregulation of extracellular matrix-related gene clusters during osteogenic differentiation of human bone marrow- and adipose tissue-derived stromal cells.

Bone marrow- and adipose tissue-derived stromal cells (BMSCs and ASCs, respectively) exhibit a similar capacity for osteogenic differentiation in vitro, but it is unclear whether they share a common differentiation process, because they originate from different tissues. The aim of this study was to explore BMSC and ASC osteogenic differentiation by focusing on the expression of extracellular matrix-related genes (ECMGs), which play a crucial role in osteogenesis and bone tissue regeneration in vivo. We characterized the gene expression profiles of BMSCs and ASCs using a custom complementary deoxyribonucleic acid microarray containing 55 ECMGs. Undifferentiated BMSCs and ASCs actively expressed a wide range of ECMGs. Once BMSCs and ASCs were placed in an osteogenic differentiation medium, 24 and 17 ECMGs, respectively, underwent considerable downregulation over the course of the culture period. The remaining genes were maintained at a similar expression level to corresponding uninduced cell cultures. Although the suppression phenomenon was consistent irrespective of stromal cell origin, collagen (COL)2A1, COL6A1, COL9A1, parathyroid hormone receptor, integrin (INT)-beta3, and TenascinX genes were only downregulated in osteogenic BMSCs, whereas COL1A2, COL3A1, COL4A1, COL5A2, COL15A1, osteopontin, osteonectin, and INT-beta1 genes were only downregulated in osteogenic ASCs. During this time period, cell viability was sustained, suggesting that the observed downregulation did not occur by selection and elimination of unfit cells from the whole cell population. These data suggest that osteogenically differentiating BMSCs and ASCs transition away from a diverse gene expression pattern, reflecting their multipotency toward a configuration specifically meeting the requirements of the target lineage. This change may serve to normalize gene expression in mixed populations of stem cells derived from different tissues.

Human adipose tissue is a source of multipotent stem cells.

Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.

In vitro microdistraction of preosteoblasts: distraction promotes proliferation and oscillation promotes differentiation.

Osteoblast biology is influenced in vivo by a 3-dimensional (3D) extracellular matrix that mediates their adhesion and interaction and by a constant state of compressive and tensile forces. To study the role of mechanical stress on osteoblasts in vitro, these parameters must be addressed. Therefore, this study describes the use of a novel, in vitro system that subjects cells to distractive and compressive forces in a 3D environment. This system, termed a microdistractor system, was used to apply linear forces to 3D collagen type I gels containing preosteoblasts. Gels were induced for up to 16 days in osteogenic medium and subjected to either constant linear distraction (distraction gels) or to repeating cycles of distraction and compression (oscillation gels). The effect of these stresses was evaluated over time by measuring proliferation rates, protein synthesis (i.e., cellular activity), and osteogenic differentiation levels. While linear forces in general appeared to increase protein synthesis, force-specific effects on proliferation and differentiation were observed. Specifically, distraction forces appeared to enhance MC3T3 proliferation while distraction/compressive forces appeared to accelerate their osteogenic differentiation program. Therefore, these results suggest that the microdistraction system may be an appropriate in vitro system for the study of mechanobiology in osteoblast phenotype.

A novel oxysterol promotes bone regeneration in rabbit cranial bone defects.

Bone morphogenetic proteins (BMPs) have played a central role in the development of regenerative therapies for bone reconstruction. However, the high cost and side-effect profile of BMPs limits their broad application. Oxysterols, naturally occurring products of cholesterol oxidation, are promising osteogenic agents alternative to BMPs. The osteogenic capacity of these non-toxic and relatively inexpensive molecules has been documented in rodent models. We studied the impact of Oxy49, a novel oxysterol analogue, on the osteogenic differentiation of rabbit bone marrow stromal cells (BMSCs). Moreover, we evaluated the capacity for in vivo bone regeneration with Oxy49 in rabbit cranial bone defects. We found that rabbit BMSCs treated with Oxy49 demonstrated differentiation along osteogenic pathways, and that complete bone regeneration occurred when cranial defects were treated with Oxy49. Collectively, these results demonstrate that Oxy49 has the ability to induce osteogenic differentiation in rabbit BMSCs with an efficacy comparable to that of BMP-2 and to promote significant bone regeneration in cranial defects. Oxysterols may be a viable novel agent in bone tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.

Natural Killer Cells Differentiate Human Adipose-Derived Stem Cells and Modulate Their Adipogenic Potential.

BACKGROUND: Natural killer cells are thought to represent more than 30 percent of all lymphocytes within the stromal vascular fraction of lipoaspirates. However, their physiologic interaction with adipocytes and their precursors has never been specifically examined. The authors hypothesized that natural killer cells, by means of cytokine secretion, are capable of promoting the differentiation of adipose-derived stem cells. METHODS: Human natural killer cells purified from healthy donors' peripheral blood mononuclear cells were activated with a combination of interleukin-2 and anti-CD16 monoclonal antibody; natural killer cell supernatant was collected. Adipose-derived stem cells isolated from raw human lipoaspirates from healthy patients were treated with growth media, growth media with natural killer cell supernatant, adipogenic media, and adipogenic media with natural killer cells supernatant. Flow cytometric analysis was performed on cells using antibodies against B7H1, CD36, CD44, CD34, CD29, and MHC-1. Adipogenic-related gene expression (PPAR-γ, LPL, GPD-1, and aP2) was assessed. Oil Red O staining was performed as a functional assay of adipocyte differentiation and adipogenesis. RESULTS: Adipose-derived stem cells maintained in growth media with natural killer cell supernatant lost markers of "stemness," including CD44, CD34, and CD29; and expressed markers of differentiation, including B7H1 and MHC-1. Adipose-derived stem cells treated with natural killer cell supernatant accumulated small amounts of lipid after 10 days of natural killer cell supernatant treatment. Adipose-derived stem cells treated with natural killer cell supernatant showed altered expression of adipogenesis-associated genes compared with cells maintained in growth media. Adipose-derived stem cells maintained in adipogenic media with natural killer cell supernatant accumulated less lipid than those cells in adipogenic media alone. CONCLUSIONS: The authors demonstrate that, through secreted factors, natural killer cells are capable of differentiating adipose-derived stem cells. In cells maintained in adipogenic media, treatment with natural killer cell supernatant modulated adipogenic potential.

Development of chemotactic smart scaffold for use in tissue regeneration.

BACKGROUND: Regenerative medicine aims to obviate the need for autologous grafting through the use of bioengineered constructs that combine stem cells, growth factors, and biocompatible vehicles. Human mesenchymal stem cells and vascular endothelial growth factor (VEGF) have both shown promise for use in this context, the former because of their pluripotent capacity and the latter because of its chemotactic activity. The authors harnessed the regenerative potential of human mesenchymal stem cells and VEGF to develop a chemotactic scaffold for use in tissue engineering. METHODS: Human mesenchymal stem cells were transduced with human VEGF via lentivirus particles to secrete VEGF. The chemotactic activity of the VEGF-transduced stem cells was evaluated via a trans-well assay. Migration through semipermeable membranes was significantly greater in chambers filled with medium conditioned by VEGF-transduced cells. VEGF-transduced cells were then seeded on apatite-coated poly(lactic-co-glycolic acid) scaffolds, thereby creating the Smart Scaffold. To determine in vivo angiogenesis, the Smart Scaffolds were implanted into subcutaneous pockets in the backs of nude mice. RESULTS: Significantly larger numbers of capillaries were observed in the Smart Scaffold compared with control implants on immunohistologic studies. For the chemotactic in vivo study, human mesenchymal stem cells tagged with a fluorescent dye (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide) were injected intravenously via tail vein after the subcutaneous implantation of the Smart Scaffolds. In vivo fluorescent imaging revealed that fluorescent dye-tagged human mesenchymal stem cells successfully accumulated within the Smart Scaffolds. CONCLUSION: These observations suggest that VEGF may play a vital role in the design of clinically relevant tissue regeneration graft substitutes through its angiogenic effects and ability to chemoattract mesenchymal stem cells.

Adult stem cell therapy for the heart.

The purpose of this review is to summarize current data leading to and arising from recent clinical application of cellular therapy for acute myocardial infarct (heart attack) and congestive heart failure. We specifically focus on use of adult stem cells and compare and contrast bone marrow and adipose tissue; two different sources from which stem cells can be harvested in substantial numbers with limited morbidity. Cellular therapy is the latest in a series of strategies applied in an effort to prevent or mitigate the progressive and otherwise irreversible loss of cardiac function that frequently follows a heart attack. Unlike surgical, pharmacologic, and gene transfer approaches, cellular therapy has the potential to restore cardiac function by providing cells capable of regenerating damaged myocardium and/or myocardial function. Skeletal muscle myoblast expansion and transfer allows delivery of cells with contractile function, albeit without any evidence of cardiomyogenesis or electrical coupling to remaining healthy myocardium. Delivery of endothelial progenitor cells (EPCs) which drive reperfusion of infarct zone tissues is also promising, although this mechanism is directed at halting ongoing degeneration rather than initiating a regenerative process. By contrast, demonstration of the ability of adult stem cells to undergo cardiomyocyte differentiation both in vitro and in vivo suggests a potential for regenerative medicine. This potential is being examined in early clinical studies.

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.

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.