Adult multipotent stromal cell cryopreservation: Pluses and pitfalls.

Study and clinical testing of adult multipotent stromal cells (MSCs) are central to progressive improvements in veterinary regenerative medicine. Inherent limitations to long-term culture preclude use for storage. Until cell line creation from primary isolates becomes routine, MSC stasis at cryogenic temperatures is required for this purpose.

Clinical Safety of Stromal Vascular Fraction Separation at the Point of Care.

Introduction: Pluripotential cells in adipose tissue may be important in long-term volume retention and regenerative effects of fat grafting. Unfortunately, graft harvest with lipoaspiration significantly depletes the population of stromal vascular cells, which includes adipose stem cells. Stromal vascular fraction (SVF) cells may be isolated from excess lipoaspirate at the point of care and used to replenish fat grafts, a technique termed cell-assisted lipotransfer (CAL).

Adipose-derived stem cells: methods for isolation and applications for clinical use.

Adipose tissue sciences have rapidly expanded since the identification of regenerative cells contained within the stromal vascular fraction (SVF) of fat. Isolation of the SVF, containing adipose-derived stem cells (ADSC), can be accomplished efficiently in the operating room or in the laboratory through enzymatic digestion of the adipose tissue and concentration of SVF. Cells can be directly re-injected as a mesotherapeutic agent, recombined with a tissue scaffold (e.

The Celution System: Automated Processing of Adipose-Derived Regenerative Cells in a Functionally Closed System.

To develop a closed, automated system that standardizes the processing of human adipose tissue to obtain and concentrate regenerative cells suitable for clinical treatment of thermal and radioactive burn wounds. A medical device was designed to automate processing of adipose tissue to obtain a clinical-grade cell output of stromal vascular cells that may be used immediately as a therapy for a number of conditions, including nonhealing wounds resulting from radiation damage. The Celution System reliably and reproducibly generated adipose-derived regenerative cells (ADRCs) from tissue collected manually and from three commercial power-assisted liposuction devices.

Acute adipocyte viability after third-generation ultrasound-assisted liposuction.

Background: Although clinical evidence of successful autologous fat transfer (AFT) using third-generation ultrasound-assisted liposuction (UAL) is readily available, no study has quantified adipocyte viability using standardized methods.

Objectives: The authors assess acute adipocyte viability following fat aspiration as a first step in determining the overall efficacy of using third-generation UAL for AFT.

Methods: Lipoaspirate samples were collected from patients who underwent elective liposuction procedures at multiple surgery centers.

Comparison of three different fat graft preparation methods: gravity separation, centrifugation, and simultaneous washing with filtration in a closed system.

Background: Successful long-term volume retention of an autologous fat graft is problematic. The presence of contaminating cells, tumescent fluid, and free lipid in the graft contributes to disparate outcomes. Better preparation methods for the fat graft before transplantation may significantly improve results.

Automated isolation and processing of adipose-derived stem and regenerative cells.

The popularity of nonhematopoietic, adult tissue-derived stem and progenitor cells for use as a cellular research tool, and ultimately as a clinical therapeutic, has increased exponentially over the past decade. Almost all adult-derived stem/progenitor cells (autologous and allogeneic), with one exception, require at least some ex vivo expansion or further manipulation prior to use to satisfy efficacy and safety requirements for preclinical or clinical use. The principal reason is the relatively low frequency of these therapeutically valuable cells within any given adult tissue, except for adipose tissue, which has been shown to have at least two log greater concentrations of these progenitor cells.

Identification and characterization of adult stem cells from human orbital adipose tissue.

Purpose: To identify pluripotential stem cells from human orbital adipose depots.

Methods: Pluripotential adipose-derived stem cells were isolated from human orbital adipose during routine blepharoplasty surgery. Fresh adipose tissue was separated in nasal fat and central (preaponeurotic) fat.

Effect of vitamin D pretreatment of human mesenchymal stem cells on ectopic bone formation.

Adult mesenchymal stem cells (MSCs) are used in contemporary strategies for tissue engineering. The MSC is able to form bone following implantation as undifferentiated cells adherent to hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds. Previous investigators have demonstrated that human MSCs (hMSCs) can be differentiated to osteoblasts in vitro by the inclusion of vitamin D and ascorbic acid.

Multipotential differentiation of adipose tissue-derived stem cells.

Tissue engineering offers considerable promise in the repair or replacement of diseased and/or damaged tissues. The cellular component of this regenerative approach will play a key role in bringing these tissue engineered constructs from the laboratory bench to the clinical bedside. However, the ideal source of cells still remains unclear and may differ depending upon the application.

Clonal analysis of the differentiation potential of human adipose-derived adult stem cells.

Pools of human adipose-derived adult stem (hADAS) cells can exhibit multiple differentiated phenotypes under appropriate in vitro culture conditions. Because adipose tissue is abundant and easily accessible, hADAS cells offer a promising source of cells for tissue engineering and other cell-based therapies. However, it is unclear whether individual hADAS cells can give rise to multiple differentiated phenotypes or whether each phenotype arises from a subset of committed progenitor cells that exists within a heterogeneous population.

Prior injury accelerates subsequent wound closure in a mouse model of regeneration.

Tissue regeneration and scarless healing involves the complete replacement and functional restoration of damaged organs and tissues. In this study of the "scarless healing" MRL mouse model, we demonstrate that 2-mm diameter through-and-through holes made in the cartilaginous part of previously injured MRL mouse ears are closed more efficiently, and that the regenerative repair response is significantly accelerated compared with unprimed MRL and control "nonhealer" strains of mice. Accelerated healing was detected both locally and distally from the original site of injury indicating the involvement of systemic components such as circulating cell types or soluble factors.

Human adipose-derived adult stem cells produce osteoid in vivo.

Adult subcutaneous fat tissue is an abundant source of multipotent cells. Previous studies from our laboratory have shown that, in vitro, adipose-derived adult stem (ADAS) cells express bone marker proteins including alkaline phosphatase, type I collagen, osteopontin, and osteocalcin and produce a mineralized matrix as shown by alizarin red staining. In the current study, the ADAS cell ability to form osteoid in vivo was determined.

Yield of human adipose-derived adult stem cells from liposuction aspirates.

Background: Primary cultures of isolated human adipose-derived adult stem (ADAS) cells are multipotent and differentiate in vitro along the adipocyte, chondrocyte, neuronal, osteoblast, and skeletal muscle pathways.

Methods: We examined the ADAS cell yield per unit volume of liposuction tissue, and their surface protein phenotype by flow cytometry. Adipogenesis was assessed by Oil Red O staining and ELISA analysis of leptin secretion.

Regulation of osteoprotegerin production by androgens and anti-androgens in human osteoblastic lineage cells.

Background: Estrogens and androgens have anti-resorptive effects on bone, although recent evidence indicates that, even in men, estrogen is the dominant sex steroid regulating bone resorption. The receptor activator of NF-kappaB ligand is essential for osteoclastic bone resorption, and its effects are blocked by the decoy receptor, osteoprotegerin (OPG). While estrogen has been shown to induce osteoblastic OPG production, the effects of androgens on OPG production have not been defined.

Neurogenic differentiation of murine and human adipose-derived stromal cells.

The identification of cells capable of neuronal differentiation has great potential for cellular therapies. We examined whether murine and human adipose-derived adult stem (ADAS) cells can be induced to undergo neuronal differentiation. We isolated ADAS cells from the adipose tissue of adult BalbC mice or from human liposuction tissue and induced neuronal differentiation with valproic acid, butylated hydroxyanisole, insulin, and hydrocortisone.

Assessment of gene regulation by bone morphogenetic protein 2 in human marrow stromal cells using gene array technology.

Marrow stromal cells can differentiate into osteoblasts, adipocytes, myoblasts, and chondrocytes. Bone morphogenetic protein 2 (BMP-2) is a potent stimulator of osteoblastic differentiation, and identification of the genes regulated by BMP-2 in these cells should provide insight into the mechanism(s) of osteoblastic differentiation. Thus, we used a conditionally immortalized human marrow stromal cell line (hMS) and a gene expression microarray containing probes for a total of 6800 genes to compare gene expression in control and BMP-2-treated cultures.

Differentiation of human marrow stromal precursor cells: bone morphogenetic protein-2 increases OSF2/CBFA1, enhances osteoblast commitment, and inhibits late adipocyte maturation.

Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor in bone marrow stroma is poorly understood, we assessed effects of bone morphogenetic protein-2 (BMP-2) on a conditionally immortalized human marrow stromal cell line, hMS(2-6), which is capable of differentiation to either lineage. BMP-2 did not affect hMS(2-6) cell proliferation but enhanced osteoblast differentiation as assessed by a 1.8-fold increase in expression of OSF2/CBFA1 (a gene involved in commitment to the osteoblast pathway), by increased mRNA expression and protein secretion for alkaline phosphatase (ALP), type I procollagen and osteocalcin (OC) (except for OC protein), and by increased mineralized nodule formation.

Effects of gonadal and adrenal androgens in a novel androgen-responsive human osteoblastic cell line.

While androgens have important skeletal effects, the mechanism(s) of androgen action on bone remain unclear. Current osteoblast models to study androgen effects have several limitations, including the presence of heterogeneous cell populations. In this study, we examined the effects of androgens on the proliferation and differentiation of a novel human fetal osteoblastic cell line (hFOB/AR-6) that expresses a mature osteoblast phenotype and a physiological number (approximately 4,000/nucleus) of androgen receptors (AR).

Development and characterization of conditionally immortalized osteoblast precursor cell lines from human bone marrow stroma.

Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312-324, 1996).

Development and characterization of a conditionally immortalized human osteoblastic cell line stably transfected with the human androgen receptor gene.

Androgens have significant beneficial effects on the skeleton. However, studies on the effects of androgens on osteoblasts are limited due to the absence of appropriate model systems that combine completeness of the osteoblastic phenotype, rapid proliferation rate, and stable expression of the androgen receptor (AR). Thus, we stably transfected the conditionally immortalized human fetal osteoblastic cell line (hFOB) with the human wild-type AR (hAR) cDNA.

Tissue cytokine patterns in patients with polymyalgia rheumatica and giant cell arteritis.

Objective: To analyze temporal artery specimens from patients with giant cell arteritis and polymyalgia rheumatica for the presence of inflammatory cytokines and to ascertain whether a specific cytokine pattern exists for the two conditions.

Design: Case series of patients having temporal artery biopsy procedures.

Setting: The outpatient clinic and the research laboratories of the Division of Rheumatology, Mayo Clinic.

HLA-DRB1 alleles in polymyalgia rheumatica, giant cell arteritis, and rheumatoid arthritis.

Objective: Immunogenetic analysis has demonstrated that giant cell arteritis (GCA) and rheumatoid arthritis (RA) are associated with 2 different domains of the HLA-DR4 molecule. The present study was undertaken to evaluate whether polymyalgia rheumatica (PMR) immunogenetically resembles GCA or RA and to determine whether expression of HLA-DRB1 alleles can be used to detect heterogeneity among PMR patients.

Methods: Forty-six patients with PMR, 52 with GCA, 122 with seropositive RA, and 72 normal individuals were genotyped for HLA-DRB1 alleles by allele-specific amplification and subsequent oligonucleotide hybridization.