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Hand dysfunction is common in systemic sclerosis (SSc). We undertook this study to evaluate the capacity of autologous adipose-derived regenerative cells (ADRCs) to improve hand function in SSc patients.The Scleroderma Treatment with Celution Processed Adipose Derived Regenerative Cells Trial was a prospective, randomized, double-blind trial of ADRCs, in which ADRCs were obtained from patients with SSc by small-volume adipose tissue harvest, and the fingers of each patient were injected with ADRCs. The primary end point was change in hand function at 24 and 48 weeks, assessed using the Cochin Hand Function Scale (CHFS). One of the secondary end points included the change in Health Assessment Questionnaire disability index (HAQ DI) at 48 weeks. Separate prespecified analyses were performed for patients with diffuse cutaneous SSc (dcSSc) and those with limited cutaneous SSc (lcSSc).Eighty-eight patients were randomized to receive ADRCs (n = 48 [32 patients with dcSSc and 16 with lcSSc]) or placebo (n = 40 [19 patients with dcSSc and 21 with lcSSc]). Change in hand function according to CHFS score was numerically higher for the ADRC group compared to the placebo group but did not achieve statistical significance (mean ± SD improvement in the CHFS score at 48 weeks 11.0 ± 12.5 versus 8.9 ± 10.5; P = 0.299). For patients with dcSSc, the between-group difference in the CHFS at 48 weeks was 6.3 points (nominal P = 0.069). For the secondary end point, the dcSSc group exhibited a between-group difference of 0.17 points in the HAQ DI (nominal P = 0.044) at 48 weeks. Of the ADRC-treated patients with dcSSc, 52% reported improvement greater than the minimum clinically important difference for both CHFS and HAQ DI compared to 16% in the placebo group (nominal P = 0.016). Small-volume adipose tissue harvest and ADRC treatment were well tolerated.While the primary end point of this trial was not achieved, efficacy trends were observed in patients with dcSSc. Adipose tissue harvest and ADRC injection were demonstrated to be feasible. Further clinical trials of this intervention in the setting of dcSSc are warranted.

Objective A number of studies have reported that application of autologous adipose-derived cell populations leads to improved outcome in different preclinical models of thermal burn injury. However, these studies were limited to assessment of relatively small injuries amounting to only ∼2% of total body surface area (TBSA) in which the complications associated with large burns (e.g.: systemic inflammation and the need for fluid resuscitation) are absent. In anticipation of translating this approach to a clinical trial in which these complications would be present we applied a preclinical model that more closely resembles a patient with large thermal burn injury requiring skin grafting. Thus, the present study used a porcine model to investigate safety and efficacy of intravenous delivery of ADRCs in the treatment of a complex burn injury comprising ∼20% TBSA and including both moderately deep (44%) partial and full thickness burns, and the injury associated with skin graft harvest. Methods Two pairs of full thickness and partial thickness burns involving in total ∼20% TBSA were created on the back of Yorkshire pigs (n = 15). Three days post-burn, full thickness wounds were excised and grafted with a 3:1 meshed autologous split thickness skin graft (STSG). Partial thickness wounds were not treated other than with dressings. Animals were then randomized to receive intravenous delivery of ADRCs (n = 8) or vehicle control (n = 7). Safety was assessed by monitoring systemic parameters (blood gases, hematology, and clinical chemistry) throughout the course of the study. Wound healing for both types of burn wound and for the skin graft donor sites was followed for 18 days using wound imaging, histology, and trans-epidermal water loss (TEWL; skin barrier function assessment). Results No serious adverse events related to ADRC infusion were noted in any of the animals. Delivery of ADRCs appeared to be safe with none of the systemic safety parameters worsened compared to the control group. TEWL and histological analyses revealed that ADRC treatment was associated with significantly accelerated healing of skin graft (27.1% vs. 1.1% on Day 5 post-grafting), donor site (52.8% vs. 33.1% on Day 5 post-excision) and partial thickness burn (81.8% vs. 59.8% on Day 18 post-treatment). Data also suggested that ADRC treatment improved parameters associated with skin graft elasticity. Conclusions This study demonstrated that intravenous delivery of autologous ADRCs appears to be a safe and feasible approach to the treatment of large burns and supports the use of ADRCs as an adjunct therapy to skin grafting in patients with severe burns.

Objective: The use of noncultured autologous stromal vascular fraction or clinical grade adipose-derived regenerative cells (ADRCs) is a promising strategy to promote wound healing and tissue repair. Nevertheless, issues regarding the optimal mode of administration remain unclear. The purpose of this study was to compare the effects of local injection and topical spray delivery of ADRCs in a porcine model of thermal burns. Approach: Full-thickness thermal burns were created on the dorsum of 10 Gottingen minipigs. Two days following injury, wounds underwent fascial excision and were randomized to receive control vehicle or freshly isolated autologous ADRCs delivered by either multiple injections into or surrounding the wound bed, or by spray onto the wound surface (0.25 × 106 viable cells/cm2). Healing was evaluated by planimetry, histopathology, and immunohistochemistry at day 7, 12, 16, 21, and 28 posttreatment. Results: In vitro analysis demonstrated that there was no substantial loss of cell number or ...

Objective: 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. Approach: 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. Results: The Celution® System reliably and reproducibly generated adipose-derived regenerative cells (ADRCs) from tissue collected manually and from three commercial power-assisted liposuction devices. The entire process of introducing tissue into the system, tissue washing and proteolytic digestion, isolation and concentration of the nonadipocyte nucleated cell fraction, and return to the patient as a wound therapeutic, can be achieved in approximately 1.5 h. An alternative approach that applies ultrasound energy ...

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. Methods Subcutaneous fat from 22 donors was divided and processed using various graft preparation methods: (1) no manipulation control, (2) gravity separation, (3) Coleman centrifugation, and (4) simultaneous washing with filtration using a commercially available system (Puregraft; Cytori Therapeutics, Inc., San Diego, Calif.). Fat grafts from various preparation methods were examined for free lipid, aqueous liquid, viable tissue, and blood cell content. Adipose tissue viability was determined by measuring glycerol release after agonist induction of lipolysis. Results All test graft preparation methods exhibited significantly less aqueous fluid and blood cell content compared with the control. Grafts prepared by washing with filtration exhibited significantly reduced blood cell and free lipid content, with significantly greater adipose tissue viability than other methods. Conclusion Washing with filtration within a closed system produces a fat graft with higher tissue viability and lower presence of contaminants compared with grafts prepared by alternate methods.

Adipose derived regenerative cells (ADRCs) are a heterogeneous population of cells including multipotent adipose derived stems cells, other progenitor cells, fibroblasts, T-regulatory cells, and macrophages. Preclinical data exist supporting benefits that are predominantly through angiogenesis, modulation of inflammation, and wound remodeling. Such effects are likely paracrine in nature. The application of autologous ADRCs has been investigated across multiple therapeutic areas. While there are numerous publications, there is a relative lack of double-blind, well-controlled, randomized clinical trials in the literature. Nevertheless, a consistency in outcomes and a consistency with preclinical and laboratory studies suggests a true positive effect. The therapeutic areas reported include orthopedics, autoimmune disease, wounds and reconstruction, cardiology, peripheral vascular disease, genitourinary disorders, gastrointestinal fistulas, and neurology. Case reports have documented wound healing in otherwise intractable wounds such as ischemic- and radiation-related cutaneous ulcers and enterocutaneous fistulas. An open label, 12-patient-study indicated substantial improvement in hand manifestations of scleroderma across multiple endpoints. Post-radical prostatectomy urinary incontinence improved in a study of 11 patients with local delivery of ADRCs. Small studies of intramyocardial delivery have been associated with trends towards benefit. The studies also indicate that same day fat harvest through liposuction, cell processing, and cell delivery is feasible and can be performed with an acceptable safety profile. The objective of this review is to highlight the interest, potential, and trends that support the need to continue evaluation and exploration for the role of ADRCs as a therapeutic agent.

Purpose: To develop an approach that models the cutaneous healing that occurs in a patient with full thickness thermal burn injury complicated by total body radiation exposure sufficient to induce sub-lethal prodromal symptoms. An assessment of the effects of an autologous cell therapy on wound healing on thermal burn injury with concomitant radiation exposure was used to validate the utility of the model.Methods: Gottingen minipigs were subjected to a 1.2 Gy total body irradiation by exposure to a 6 MV X-ray linear accelerator followed by ∼10 cm2 full thickness burns (pre-heated brass block with calibrated spring). Three days after injury, wounds were excised to the underlying fascia and each animal was randomized to receive treatment with autologous adipose-derived regenerative cells (ADRC) delivered by local or intravenous injection, or vehicle control. Blood counts were used to assess radiation-induced marrow suppression. All animals were followed using digital imaging to assess wound healing....

Background. Acute kidney injury (AKI) represents a major clinical problem with high mortality and limited causal treatments. The use of cell therapy has been suggested as a potential modality to improve the course and outcome of AKI. Methods. We investigated the possible renoprotection of freshly isolated, uncultured adipose tissue-derived stem and regenerative cells (ADRCs) before and after cryopreservation in a rat ischemia–reperfusion (I–R) model of AKI. Results. We demonstrated that ADRC therapy drastically reduced mortality (survival 100% vs. 57%, ADRC vs. controls, respectively) and significantly reduced serum creatinine (sCr on Day 3: 3.03 ± 1.58 vs. 7.37 ± 2.32 mg/dL, ADRC vs. controls, respectively). Histological analysis further validated a significantly reduced intratubular cast formation, ameliorated acute tubular epithelial cell necrosis and mitigated macrophage infiltration. Furthermore, a reduced RNA expression of CXCL2 and IL-6 was found in the ADRC group which could explain the reduced macrophage recruitment. Use of cryopreserved ADRCs resulted in an equally high survival (90% vs. 33% in the control group) and similarly improved renal function (sCr on Day 3: 4.64 ± 2.43 vs. 7.24 ± 1.40 mg/dL in controls). Conclusions. Collectively, these results suggest a potential clinical role for ADRC therapy in patients with AKI. Importantly, cryopreservation of ADRCs could offer an autologous treatment strategy for patients who are at high risk for AKI during planned interventions.

Current practice of autologous fat transfer for soft tissue augmentation is limited by poor long-term graft retention. Adipose-derived regenerative cells (ADRCs) contain several types of stem and regenerative cells, which may help improve graft retention through multiple mechanisms. Using a murine fat transplantation model, ADRCs were added to transplanted fat to test whether ADRCs could improve the long-term retention of the grafts. This study showed, at both 6 and 9 months after transplantation, ADRCs not only increased graft retention by 2-fold but also improved the quality of the grafts. ADRC-supplemented grafts had a higher capillary density, indicating ADRCs could promote neovascularization. Further cell tracking and gene expression studies suggest ADRCs may promote angiogenesis and adipocyte differentiation and prevent apoptosis through the expression of various growth factors, including VEGFA and IGF-1. Taken together, these results suggest a potential clinical utility of ADRCs in facilitating autologous fat transfer for soft tissue augmentation.

Adipose tissue can be harvested in large amounts with minimal morbidity. It contains numerous cells types, including adipocytes, preadipocytes, vascular endothelial cells and vascular smooth muscle cells; it also contains cells that have the ability to differentiate into several lineages, such as fat, bone, cartilage, skeletal, smooth, and cardiac muscle, endothelium, hematopoietic cells, hepatocytes and neuronal cells. Cloning studies have shown that some adipose-derived stem cells (ADSCs) have multilineage differentiation potential. ADSCs are also capable of expressing multiple growth factors, including vascular endothelial growth factor and hepatocyte growth factor. Early, uncontrolled, non-randomized clinical research, applying fresh adipose-derived cells into a cranial defect or undifferentiated ADSCs into fistulas in Crohn's disease, has shown healing and an absence of side effects. The combination of these properties, and the large quantity of cells that can be obtained from fat, suggests that this tissue will be a useful tool in biotechnology.

BACKGROUND: The goals of the Cord Blood Transplantation (COBLT) Study banking program initiated in 1996 were to develop standard operating procedures (SOPs) for cord blood (CB) donor recruitment and banking and to build an ethnically diverse unrelated CB bank to support a transplantation protocol. STUDY DESIGN AND METHODS: The program included collection centers, three banks, a steering committee, and a medical coordinating center (MCC) that developed and validated SOPs and a Web-based data collection system. External oversight was performed by the National Heart, Lung, and Blood Institute and the MCC. RESULTS: A total of 34,799 potential donors were screened and 20,710 consented. A total of 17,207 ethnically diverse units were collected between 1998 and 2001. A total of 11,077 (64%) units were cryopreserved and quarantined. Of these, 79 percent met eligibility criteria and were HLA-typed and entered into the search registry. Higher CB volumes and cell counts were obtained from cesarean sections compared to vaginal deliveries. Units from African American persons contained lower cell counts per volume compared to other ethnicities. Birth weight correlated with volume and cell content. External oversight was accomplished through custom reports generated by the data collection system and periodic site visits. During maintenance, a breach in the SOPs was detected during a site visit at one of the banks. These units were designated for future use in nonclinical research. CONCLUSION: The COBLT Study demonstrated that SOPs and data collection can be implemented in multiple banks coordinated by one MCC. Relationships between donor demographics and CB content may be useful in the development of other CB banking programs.

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.

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.

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.

During the process of normal hematopoiesis, proliferation is tightly linked to maturation. The molecular mechanisms that lead to production of mature effector cells with a variety of phenotypes and functions from a single multipotent progenitor are only beginning to be elucidated. It is important to determine how these maturation events are regulated at the molecular level, because this will provide significant insights into the process of normal hematopoiesis as well as leukemogenesis. Transcription factors containing the highly conserved homeobox motif show considerable promise as potential regulators of hematopoietic maturation events. In this study, we focused on identification and characterization of homeobox genes of the HOX family that are important in regulating normal human myeloid differentiation induced by the hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF). We have identified three homeobox genes, HOX A5, HOX B6, and HOX B7, which are expressed during early myelopoiesis. Treating bone marrow cells with antisense oligodeoxynucleotides to HOX A5 resulted in inhibition of granulocytic/monocytic hematopoiesis and increased the generation of erythroid progenitors. Also, overexpression of HOX A5 inhibited erythroid differentiation of the K562 cell line. Based on these observations, we propose that HOX A5 functions as an important regulator of hematopoietic lineage determination and maturation.

In 1995, the National Heart Lung and Blood Institute (NHLBI) solicited requests for a proposal (RFP) entitled "Transplant Centers for Clinical Research on Transplantation of Umbilical Cord Stem and Progenitor Cells." Three banks, six transplant centers, and one medical coordinating center (MCC) (Table 1) were funded with the overall goal of banking cord blood units (CBU) using a single manual of operations. Furthermore, the clinical protocols to evaluate the transplant outcome for adult and pediatric recipients of these well-characterized CBU would be analyzed in a uniform fashion. Because of the intense interest of the transplantation community in the policies and procedures for cord blood collection and processing, the principal investigators of the cord blood banks (CBB) and NHLBI elected to submit for publication the rationale and an abridged, but detailed, version of the standard operating procedures (SOP) developed between October 1996 and July 1998 prior to the initiation of the clinical protocols to be performed with these CBU. As the SOP will be refined over time, the complete SOP and subsequent amendments will be published and continually updated on the websites from the MCC-The EMMES Corporation (www.EMMES.com). All forms referred to in this document may be obtained from the EMMES website. It is hoped that the publication of this document will lay down a framework that will not only facilitate the development of other CBB but also help us more rapidly define what constitutes an "acceptable" CBU product.

Objective: 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.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that stimulates the proliferation, maturation, and functional activity of myeloid cells in peripheral blood and bone marrow. Expression of GM-CSF is tightly regulated and is limited to cells stimulated directly (T cells, macrophages) or indirectly (fibroblasts, endothelial cells) by immune challenge. Several studies of the transcriptional control of GM-CSF expression have elucidated a region of the GM-CSF promoter that mediates positive regulatory activity in a number of cell types. This region contains a direct repeat of the sequence CATTA/T that extends from nucleotides -37 to -48 upstream of the start of mRNA synthesis. Although specific DNA:protein interactions have been shown within this region, neither the nature nor the number of nuclear factors responsible for these interactions have been characterized. In this study, we use DNase I footprinting analysis to demonstrate that point mutations, which inactivate the GM-CSF promoter, disrupt DNA:protein interactions within this region. By combined electrophoretic mobility shift and ultraviolet cross-linking analysis, we have detected several protein species that bind specifically to the positive regulatory sequence.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates myeloid progenitor cell proliferation and enhances the function of terminally differentiated effector cells. Interleukin-3 (IL-3) stimulation results in the proliferation and maturation of early bone marrow progenitor cells. These activities are mediated by non-tyrosine kinase-containing receptors which consist of ligand-specific alpha subunits that complex with a common beta subunit required for signal transduction. Both GM-CSF and IL-3 rapidly and transiently induce expression of early growth response gene 1 (egr-1) in the human factor-dependent cell line TF-1. To define the mechanism of early response gene induction by GM-CSF and IL-3, growth factor- and serum-starved TF-1 cells transfected with recombinant constructs containing sequences of the human egr-1 promoter were stimulated with GM-CSF or IL-3. A 116-nucleotide (nt) region of the egr-1 promoter which contains sequences inducible by GM-CSF and IL-3 was defined. DNase I footprint analysis identified a 20-nt region, including nt -57 to -76, which contains a potential cyclic AMP (cAMP) response element (CRE). Electrophoretic mobility shift assays performed with CREB antibody confirmed the presence of CREB in the DNA-binding complex. Mutational analysis of the cytokine-responsive region of the egr-1 promoter revealed that both the cAMP response and serum response elements are required for induction by GM-CSF and IL-3. Nuclear extracts from GM-CSF- or IL-3-stimulated but not unstimulated TF-1 cells contain factors which specifically bind to the Egr-1-binding site in the nt -600 to -480 region of the promoter. Electrophoretic mobility shift assays were performed with antibodies against the Egr-1 protein to demonstrate the presence of the protein product in the shifted complex. Our studies suggest that the Egr-1 protein may further stimulate transcription of the egr-1 gene in response to GM-CSF as a secondary event.

There is increasing clinical interest focused on ex vivo manipulation and expansion of hematopoietic cells. In this study, we demonstrate that a simple combination of growth factors can expand progenitors to yield functional myeloid cells. Furthermore, this system can produce mature, functionally competent cells in the absence of fetal bovine serum (FBS), which will enhance the clinical utility of this approach. Hematopoietic progenitor cells obtained from normal bone marrow and from leukapheresis products were studied. The mononuclear fraction was enriched for CD34 cells using the Ceprate CD34 biotin kit (CellPro #LC34-1 or LC34-2). The selected cells were expanded for two weeks in Iscove's medium supplemented with 20% FBS and various combinations of interleukin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF) and interleukin-6 (IL-6) added either simultaneously or sequentially. The optimal combination of these factors identified for myeloid expansion was simultaneous addition of IL-3, SCF and G-CSF (at 50 ng/ml each), resulting in an average 773 ± 133-fold expansion of nucleated cells (n = 5). When corrected for the purity of CD34 cells in the starting population, the mean fold expansion with IL-3, SCF and G-CSF was 2,265 ± 729. A mean of 74.7 ± 10.5% (n = 3) of the expanded cells was positive for CD11b; 86-91% (n = 2) of the cells were promyelocytes or more mature granulocytes. Functional assays demonstrated normal phagocytosis and intracellular killing of Staphylococcus aureus (S. aureus) by the expanded cell population. Studies performed using cells expanded in defined serum-free media demonstrated that fold expansion was decreased and that the cells produced were less mature and functionally less competent than cells expanded with FBS. The decreased expansion could be partially reversed, and the functionality almost completely restored by the addition of autologous plasma.

As the frequency of fat grafting to the breast has increased, some investigators have raised the possibility that this procedure may potentially increase the risks associated with breast cancer. Their concerns included not only interference with cancer detection, but also promotion of tumor formation or recurrence mediated by mechanisms such as aromatase expression, angiogenesis, and tumor stromal cells. However, published clinical studies describing outcomes of fat grafting to the breast in more than 2000 patients have not reported any increase in new or recurrent cancers. The reason for this apparent disconnect may lie in the small sample sizes and relatively short follow-up, but it may also reside in the considerable gap between laboratory studies or theoretical considerations suggesting potential risks and the actual clinical practice. This review discusses potential risks of current and novel approaches to autologous fat grafting to the breast within the context of both the underlying science and clinical practice.

Human adipose tissue has been shown to contain a population of cells that possesses extensive proliferative capacity and the ability to differentiate into multiple cell lineages. These cells are referred to as adipose tissue-derived stem cells (ADSCs) and are generally similar, though not identical, to mesenchymal stem cells (also referred to as marrow stromal cells). ADSCs for research are most conveniently extracted from tissue removed during an elective cosmetic liposuction procedure but may also be obtained from resected adipose tissue. This chapter describes surgical procedures associated with improved ADSC recovery and the processes by which aspirated adipose tissue is washed and digested with collagenase to yield a heterogeneous population from which ADSCs can be expanded. The large volume of tissue obtained from a liposuction procedure (average approximately 2 L), combined with the relatively high frequency of ADSC within the digestate, yields substantially more stem cells than can be realized from marrow without extensive expansion in culture.

Adipose tissue consists of mature adipocytes and a mononuclear cell fraction termed adipose tissue-derived cells (ADCs). Within these heterogeneous ADCs exists a mesenchymal stem cell-like cell population, termed adipose tissue-derived stem cells. An important clinical advantage of adipose tissue-derived stem cells over other mesenchymal stem cell populations is the fact that they can be isolated in real time in sufficient quantity, such that ex vivo expansion is not necessary to obtain clinically relevant numbers for various therapeutic applications.The aim of this investigation was to evaluate the therapeutic potential of freshly isolated ADCs in treating rats acutely following myocardial infarction. Rats underwent 45 min of left anterior descending artery occlusion followed by reperfusion. Fifteen minutes post-myocardial infarction, saline or 5 x 10(6) ADCs from green fluorescent protein-expressing transgenic rats were injected into the chamber of the left ventricle. Left ventricular function and morphometry was followed with 2-D echocardiography for 12 wk, at which point hearts were harvested for histological analysis.Twelve weeks following cell therapy, left ventricular end-diastolic dimension was less dilated while the ejection fraction and cardiac output of ADC-treated rats were significantly improved compared to control rats (P0.01). Despite this benefit, absolute engraftment rates were low. This paradox may be partially explained by ADC-induced increases in both capillary and arteriole densities.These data confirm the therapeutic benefit of freshly isolated ADCs delivered post-MI and suggest a novel beneficial mechanism for ADCs through a potent proangiogenic effect.

Human adipose tissue has been shown to contain a population of cells that possesses extensive proliferative capacity and the ability to differentiate into multiple cell lineages. These cells are referred to as adipose tissue-derived stem cells (ADSCs) and are generally similar, though not identical, to mesenchymal stem cells (also referred to as marrow stromal cells). ADSCs for research are most conveniently extracted from tissue removed during an elective cosmetic liposuction procedure but may also be obtained from resected adipose tissue. This chapter describes surgical procedures associated with improved ADSC recovery and the processes by which aspirated adipose tissue is washed and digested with collagenase to yield a heterogeneous population from which ADSCs can be expanded. The large volume of tissue obtained from a liposuction procedure (average approximately 2 L), combined with the relatively high frequency of ADSC within the digestate, yields substantially more stem cells than can be realized from marrow without extensive expansion in culture.

Background Human adipose tissue has been shown to contain multipotent cells with properties similar to mesenchymal stromal cells. While there have been many studies of the biology of these cells, no study has yet evaluated issues associated with tissue harvest. Methods Adipose tissue was obtained from the subcutaneous space of the abdomen and hips of 10 donors using both syringe and pump-assisted liposuction. Tissue was digested with collagenase and then assayed for the presence of different stem and progenitor cell types using clonogenic culture assays, including fibroblast colony-forming unit (CFU-F) and alkaline phosphatase-positive colony-forming unit (CFU-AP). Paired analysis of samples obtained from the same individual was used to compare harvest method and site. Results Syringe suction provided significantly greater recovery of adipocytes and a non-significant trend towards improved recovery of cells in the adipocyte-depleted fraction. There was considerable donor-to-donor variation in stem cell recovery. However, paired analysis of tissue obtained from different subcutaneous sites in the same donor showed that tissue harvested from the hip yielded 2.3-fold more CFU-F/unit volume and a 7-fold higher frequency of CFU-AP than that obtained from the abdomen. These differences were statistically significant. Discussion Harvest site influences the stem and progenitor cell content of subcutaneous adipose tissue.

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. Current research for many applications is focused on the use of adult stem cells. The properties of adult stem cells that make them well-suited for regenerative medicine are (1) ease of harvest for autologous transplantation, (2) high proliferation rates for ex vivo expansion and (3) multilineage differentiation capacity. This review will highlight the use of adipose tissue as a reservoir of adult stem cells and draw conclusions based upon comparisons with bone marrow stromal cells. (Keio JM ed 54 (3): 132-141, September 2005)

Recent preclinical and clinical studies have suggested that adult stem cells have the ability to promote the retention or restoration of cardiac function in acute and chronic ischemia. Published clinical studies have used autologous donor cells, including skeletal muscle myoblasts, cultured peripheral blood cells, or bone marrow cells. However, our research and that of others indicates that human adipose tissue is an alternative source of cells with potential for cardiac cell therapy. These findings include the presence of cells within adipose tissue that can differentiate into cells expressing a cardiomyocytic or endothelial phenotype, as well as angiogenic and antiapoptotic growth factors. This potential is supported by preclinical studies in large animals.

Summary of Background Data. Disc degeneration is asubstantial clinical problem. Disc degeneration beginswith a loss of disc cells and alterations in the extracellularmatrix of the disc. One promising approach for this prob-lem involves the use of cells transplanted to the degen-erative disc to achieve functional tissue repair. Methods. The rationale for using cellular therapy fordisc degeneration is discussed. The basic science studiesinvolving cellular transplantation to the disc are reviewedand future directions of this line of research are dis-cussed. Results. Although substantial work remains, the futureof cellular therapies for symptomatic disc degenerationappears promising. Conclusion. Continued research is warranted to fur-ther define the optimal cell type, scaffolds, and adjuvantsthat will allow successful disc repair in human patients. Key words: disc degeneration, cell, repair, scaffold. Spine 2005;30:S14–S19 Disc degeneration is a universal part of human aging andin most cases results in minimal or self-limited symp-toms.However,thesmallsegmentofthepopulationthatexperiences disabling spinal pain thought to be in partattributable to disc degeneration represents a dispropor-tionate medical, societal, and economic challenge. Cur-rent treatment options range from medicinal inflamma-tionmanagement,toinvasiveproceduresincludingspinefusion and recently spinal arthroplasty. Unfortunately,all currently available approaches are limited to treatingthe symptoms of the degenerative process and not theunderlying biologic alterations of the disc. Recently,there has been a growing interest in developing strategiesto address the underlying biologic imbalances that leadto symptomatic disc degeneration.Thenormalintervertebraldiscissandwiched betweenupper and lower osteochondral endplates and is com-posed of a tough, outer anulus fibrosus (AF) and gelati-nous, proteoglycan-rich inner nucleus pulposus (NP),which attracts and retains water.

This is a report of a 7-year-old girl suffering from widespread calvarial defects after severe head injury with multifragment calvarial fractures, decompressive craniectomy for refractory intracranial hypertension and replantation of cryopreserved skull fragments. Chronic infection resulted in an unstable skull with marked bony defects. Two years after the initial injury the calvarial defects were repaired. Due to the limited amount of autologous cancellous bone available from the iliac crest, autologous adipose derived stem cells were processed simultaneously and applied to the calvarial defects in a single operative procedure. The stem cells were kept in place using autologous fibrin glue. Mechanical fixation was achieved by two large, resorbable macroporous sheets acting as a soft tissue barrier at the same time. The postoperative course was uneventful and CT-scans showed new bone formation and near complete calvarial continuity three months after the reconstruction.

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.

The cell surface receptor Notch1 is expressed on CD34+ hematopoietic precursors, whereas one of its ligands, Jagged1, is expressed on bone marrow stromal cells. To examine the role of Notch signaling in early hematopoiesis, human CD34+ cells were cultured in the presence or absence of exogenous cytokines on feeder layers that either did or did not express Jagged1. In the absence of recombinant growth factors, Jagged1 decreased myeloid colony formation by CD34+ cells, as well as 3H-thymidine incorporation and entry into S phase. In the presence of a strong cytokine signal to proliferate and mature, (interleukin 3 [IL-3] and IL-6, stem cell factor [SCF], and G-CSF), Jagged1 did not significantly alter either the fold expansion or the types of colonies formed by CD34+ cells. However, in the presence of SCF alone, Jagged1 increased erythroid colony formation twofold. These results demonstrate that Notch can modulate a growth factor signal, and that in the absence of growth factor stimulation, the Jagged1-Notch pathway preserves CD34+ cells in an immature state.

Human immunodeficiency virus type 1 (HIV-1)-infected individuals may exhibit multiple hematopoietic abnormalities including anemia, granulocytopenia, thrombocytopenia, and myelodysplastic/hyperplastic alterations of the bone marrow, suggesting virus-induced abnormalities in the bone marrow microenvironment (7, 9, 11, 35). Evidence of alteration of fetal hematopoiesis including leukopenia, anemia, and thrombocytopenia has also been found in aborted fetuses from HIV-1-seropositive women (6, 32). These observations suggest that HIV-1 infection may affect processes important during early stages of hematopoiesis. However, several different factors, including direct intracellular effects of virus infection, interaction with viral proteins at the cell surface, perturbation of the cytokine network, or immune-mediated effects, may play a role. Following HIV-1 infection in vitro in long-term bone marrow cultures, inhibition of hematopoietic progenitor cell production occurs and alteration of production of cytokines relevant to hematopoiesis has been documented (14, 15, 27). These infected stromal cultures showed reduced production of the cytokines interleukin-6 (IL-6) and granulocyte colony-stimulating factor, which could affect regulatory signals important in hematopoiesis. Further in vitro studies suggested that HIV-1-induced suppression of hematopoiesis is mediated by the HIV-1-encoded envelope glycoprotein gp120 and the Nef regulatory protein, as well as by cellular proteins such as tumor necrosis factor alpha (8, 23). The p24 Gag protein of HIV-1 also was shown to inhibit myeloid colony formation of bone marrow cultures but had minor effects on erythroid colony formation (29). Purified CD34+ cells were reported to be susceptible to HIV-1 infection, as shown by the presence of proviral sequences in the ensuing colonies of erythroid and myeloid lineages generated from these cells (10). These effects could be influenced by the infection of microvascular endothelial cells of bone marrow stromal cultures from HIV-seropositive patients (27). Infection of these cells could affect the relevant neighboring microenvironment, by providing a continuing source of virus and by causing alteration of local cytokine levels. Therefore, HIV is likely to alter the stromal/progenitor cell microenvironment that supports hematopoiesis. However, these previous studies on in vitro consequences of virus infection could not determine how HIV-1 infection influences complex hematopoietic microenvironments in vivo. To investigate how HIV-1 might affect hematopoiesis in vivo, we used the SCID-hu (Thy/Liv) mouse model, in which human fetal thymus and liver tissue are coimplanted into severe combined immunodeficient mice, resulting in a functional human hematopoietic organ (Thy/Liv) (24, 28). This model allows maintenance and differentiation through thymopoiesis of human hematopoietic progenitor cells (28) and also recapitulates the effects of HIV-1 infection in the human thymus. Direct infection of various strains of HIV-1 into Thy/Liv implants results in severe depletion of CD4-bearing human thymocytes which mirrors that seen in infected individuals (2, 5, 17, 20, 21, 30). Morphologic alterations of the thymic stroma, possibly due to infection of thymic epithelial cells, have been seen (30). These effects are precipitated by a large proviral burden, which may be as high as nearly one copy of the viral genome per CD4+ cell (18). Both direct virus-induced killing (18) and indirect apoptotic effects (31) have been implicated in the observed depletion of CD4+ thymocytes. Furthermore, this model has been used to determine the viral accessory genes involved in pathogenesis (3, 16) and the response of HIV-1 to antiviral therapies (19, 25, 26, 33). Previously, McCune et al. (24) reported that human pluripotent hematopoietic progenitor cells capable of giving rise to myeloid and erythroid colonies ex vivo in response to cytokines are maintained for extended periods of time in this model. The SCID-hu system has thus proven relevant to the clinical situation, is amenable to manipulation and analyses, and offers an opportunity to investigate the effects of HIV-1 infection on multiple arms of hematopoiesis. Here we report that HIV-1 infection profoundly decreases the ability to recover hematopoietic colony-forming activity (CFA) from Thy/Liv implants. However, this effect is reversible following administration of combination antiretroviral therapy. Our studies thus establish a causal effect of viral replication on hematopoiesis of multiple lineages. In addition, the reversible nature of this inhibitory effect following therapy suggests that neither the very immature hematopoietic progenitor cell nor the differentiation-inducing microenvironment is destroyed by high levels of HIV-1. Furthermore, we identify a viral strain, isolated from a pediatric patient exhibiting severe hematologic abnormalities, which preferentially inhibits hematopoietic CFA rather than inducing CD4+ thymocyte depletion. Together our studies suggest that HIV-1 may be directly responsible for many of the hematopoietic perturbations seen in infected individuals.

Human T-cell leukemia virus type I (HTLV-1) is the etiologic agent of adult T-cell leukemia and lymphoma and HTLV-1-associated myelopathy-tropical spastic paraparesis. We examined whether HTLV could productively infect human hematopoietic progenitor cells. CD34+ cells were enriched from human fetal liver cells and cocultivated with cell lines transformed with HTLV-1 and -2. HTLV-1 infection was established in between 10 and >95% of the enriched CD34+ cell population, as demonstrated by quantitative PCR analysis. HTLV-1 p19 Gag expression was also detected in infected hematopoietic progenitor cells. HTLV-1-infected hematopoietic progenitor cells were cultured in semisolid medium permissive for the development of erythbroid (BFU-E), myeloid (CFU-GM), and primitive progenitor (CFU-GEMM, HPP-CFC, or CFU-A) colonies. HTLV-1 sequences were detected in colonies of all hematopoietic lineages; furthermore, the ratio of HTLV genomes to the number of human cells in each infected colony was 1:1, consistent with each colony arising from a single infected hematopoietic progenitor cell. Severe combined immunodeficient mice engrafted with human fetal thymus and liver tissues (SCID-hu) develop a conjoint organ which supports human thymocyte differentiation and maturation. Inoculation of SCID-hu mice with HTLV-1-infected T cells or enriched populations of CD34+ cells established viral infection of thymocytes 4 to 6 weeks postreconstitution. Thymocytes from two mice with the greatest HTLV-1 proviral burdens showed increased expression of the CD25 marker and the interleukin 2 receptor alpha chain and perturbation of CD4+ and CD8+ thymocyte subset distribution profiles. Hematopoietic progenitor cells and thymuses may be targets for HTLV infection in humans, and these events may play a role in the pathogenesis associated with infection.

The human immunodeficiency virus (HIV) Rev and human T-cell leukemia virus (HTLV) Rex proteins regulate viral RNA processing. Both proteins act to overcome the block to viral structural gene expression, at least in part, by reversing the inhibitory effect of intronic RNA sequences, termed cis-acting repressive (CRS) sequences. Using HTLV type II (HTLV-II) as a model, we recently showed that the function of a 5' long terminal repeat (LTR) CRS correlates with in vitro binding by both polypyrimidine tract binding (PTB) protein (also known as hnRNP I) and hnRNP A1 to CRS RNA (1,2). Using radioimmunoprecipitation of proteins ultraviolet (UV) crosslinked to each HIV CRS RNA with monoclonal anti-hnRNP antibodies, we now demonstrate that hnRNP I and hnRNP A1 bind to two different HIV-1 CRS RNAs. In addition, we show that hnRNP I and hnRNP A1 binding to HIV-1 CRS RNAs can be specifically competed by HTLV-II CRS RNAs using electrophoretic mobility shift assay (EMSA)/UV crosslinking assays. Binding by both hnRNP I and hnRNP A1 to HIV-1 and HTLV-II CRS RNAs suggests a role for these proteins in CRS function that may be influenced by the Rev and Rex proteins, respectively.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates myeloid progenitor cell proliferation and enhances the function of terminally differentiated effector cells. Interleukin-3 (IL-3) stimulation results in the proliferation and maturation of early bone marrow progenitor cells. These activities are mediated by non-tyrosine kinase-containing receptors which consist of ligand-specific alpha subunits that complex with a common beta subunit required for signal transduction. Both GM-CSF and IL-3 rapidly and transiently induce expression of early growth response gene 1 (egr-1) in the human factor-dependent cell line TF-1. To define the mechanism of early response gene induction by GM-CSF and IL-3, growth factor- and serum-starved TF-1 cells transfected with recombinant constructs containing sequences of the human egr-1 promoter were stimulated with GM-CSF or IL-3. A 116-nucleotide (nt) region of the egr-1 promoter which contains sequences inducible by GM-CSF and IL-3 was defined. DNase I footprint analysis identified a 20-nt region, including nt -57 to -76, which contains a potential cyclic AMP (cAMP) response element (CRE). Electrophoretic mobility shift assays performed with CREB antibody confirmed the presence of CREB in the DNA-binding complex. Mutational analysis of the cytokine-responsive region of the egr-1 promoter revealed that both the cAMP response and serum response elements are required for induction by GM-CSF and IL-3. Nuclear extracts from GM-CSF- or IL-3-stimulated but not unstimulated TF-1 cells contain factors which specifically bind to the Egr-1-binding site in the nt -600 to -480 region of the promoter. Electrophoretic mobility shift assays were performed with antibodies against the Egr-1 protein to demonstrate the presence of the protein product in the shifted complex. Our studies suggest that the Egr-1 protein may further stimulate transcription of the egr-1 gene in response to GM-CSF as a secondary event.

The shift from viral regulatory to structural gene expression in human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) is mediated by Rex. We have previously shown that HTLV-II Rex acts through an element in R/U5 of the 5′ long terminal repeat (LTR), the Rex-responsive element (RxRE), and that Rex protein binds to specific RNA sequences, the Rex binding element (RBE), contained within the RxRE (Black et al., J. Virol. 65, 6645-6653, 1991b). Rex action through the RBE (nt 405-520) overcomes the inhibition of expression conferred by a contiguous LTR RNA regulatory element, which contains cis-acting repressive sequences (CRS; nt 520-630) that are not bound by Rex protein (Black et al., Virology, 181, 433-144, 1991a). We now show by electrophoretic mobility shift assay (EMSA) that cellular proteins in a HeLa nuclear extract bind specifically to RNA transcripts containing the HTLV-II CRS. Using ultraviolet (uv) crosslinking of gel-retarded bands, we identified a major protein species of approximately 60 kDa, p60CRS, that binds to CRS RNA and, with weaker affinity, to RBE RNA. In addition, a distinct 40-kDa protein, p40CRS, binds to U5 RNA (nt 645-750) downstream from the CRS. Specific deletions within CRS RNA can reduce or abrogate binding to this 60-kDa protein. EMSA and uv crosslinking assays also suggest that both p60CRS and p40CRS interact with CRS RNA. CRS function in a 5′ LTR-linked gene expression assay correlates with the ability of both p60CRS and p40CRS to interact with 5′ LTR RNA in vitro.

The hematopoietic growth factor GM-CSF (granulocyte-macrophage colony-stimulating factor) is expressed by activated but not resting T lymphocytes. Previously, we localized GM-CSF-inducible promoter activity to a 90-bp region of GM-CSF 5'-flanking sequences extending from bp -53 to +37. To more precisely identify the GM-CSF DNA sequences required for inducible promoter activity in T lymphocytes, we have performed mutagenesis within a region of GM-CSF 5'-flanking sequences (bp -57 to -24) that contains the repeated sequence CATT(A/T). Mutations that do not alter the repeated CATT(A/T) sequence do not eliminate inducible promoter activity, whereas mutation or deletion of either of the CATT(A/T) repeats eliminates all inducible promoter activity in T-cell lines and in primary human T lymphocytes. Thus, both copies of the direct repeat CATT(A/T) are required for mitogen-inducible expression of GM-CSF in T cells.

We and others have previously demonstrated that unrelated cyropreserved banked CB is an effective alternative source of allogeneic stem cells but is limited by delays in myeloid and platelet engraftment and immune reconstitution (Kurtzberg et al, NEJM 1996; Cairo et al, Blood 1997). CB nucleated (nuc) cell count, nucleated RBC and total CFU have been previously demonstrated to be associated with time to hematopoietic reconstitution (Rubinstein et al, NEJM 1998; Stevens et al, Blood 2002). More recently CB CD34+ content has been demonstrated to be significantly correlated to speed of engraftment and survival (Wagner et al, Blood 2002; Styczynski et al, BMT 2004). CB CD34+ subsets (CD38−, CD61+, CD90+) may also be critically important in predicting time to CB hematopoietic reconstitution, ex-vivo CB expansion and/or CB gene therapy. We investigated the relationships of CB CD34+ and CD34+ subsets with CB total CFU, CFU subsets, nuc cell, nuc RBC, ethnicity, gender, birth weight (BWT), gestational age (GA) and type of delivery in over 8,000 CB units. Briefly, CB was collected and processed (Fraser et al, J Hematother 1998) and aliquots were obtained for flow cytometry and in-vitro colony assays. Statistical analysis was based on multiple regression analysis and analysis or variance of logged counts. Approximately 35,000 maternal donors were screened, 20,700 consented, 16,700 CB collected and 8,730 CB units entered long-term cryopreserved storage. Ethnic distribution was 42% Caucasian, 21% Hispanic, 14% African-American, 11% Asian and 12% mixed. CB was collected by vaginal delivery (74%), at 39–40 weeks GA (61%) and 3–4000 Gm BWT (73%). Mean ± SD CD34+ and CD34+/38−, 61+, 90+ were 34±42, 3.4±4.8, 7.3±9.5 and 0.3±1.9 x 105, respectively. Mean SD of total CFU, BFU-E, CFU-GM and CFU-GEMM was 23.2±19.8, 13.3±3.1, 9.3±8.6 and 0.5±1.1, respectively. There was a significant correlation of log CD34+ with log nuc cell count and log nuc RBC count (R= 0.64, p

Several cell surface and cytoplasmic markers specific for the megakaryocyte-platelet lineage have been described. However, as yet, none of these has been shown to be expressed on cells earlier than the committed megakaryocyte progenitor, CFU-Meg. The present study was aimed at determining whether platelet lineage antigens could be detected on human pluripotential stem cells. Rabbit antiserum against human platelets (APS) was extensively absorbed with erythrocytes and either platelets, neutrophils, monocytes, or cells of the monocytic cell line U937. The anti-stem cell antibodies in each absorbed antiserum were determined using a complement-dependent cytotoxic assay for the pluripotential stem cell CFU-mix. Platelets alone removed anti- stem cell antibodies from APS. Absorption of APS with platelets from a patient with Glanzmann's thrombasthenia failed to remove the anti-stem cell activity, providing evidence for involvement of the platelet glycoprotein IIb/IIIa complex. Antiserum against purified glycoprotein IIb and against glycoprotein IIIa also recognized stem cells, resulting in reduced formation of mixed colonies. Absorption of these antisera with normal platelets removed the anti-stem cell activity, indicating that both IIb and IIIa are represented on stem cells. Hence, cell surface antigens specific for the stem cell-megakaryocyte-platelet pathway are expressed on the platelet glycoprotein IIb/IIIa complex.

Erythroid differentiation is mediated by several interacting factors which include the glycoprotein hormone erythropoietin (Epo), interleukin-3 (IL-3) in the mouse, and erythroid-potentiating activity (EPA) in humans. Each of these factors binds to specific cell surface receptors on responsive target cells, but the way in which these factors interact to modulate erythropoiesis is unknown. In the present study, we used the human erythroleukemic cell line K562 to examine expression and regulation of the receptor for Epo using 125I-labeled, bioactive recombinant human Epo. K562 cells expressed low numbers of a single class of high-affinity Epo receptors corresponding to 4 to 6 receptors per K562 cell (KD = 270 to 290 pmol/L). Treatment of K562 cell cultures with medium conditioned by the EPA-secreting cell line U937 (U937CM) increased receptor expression 2.6 to 3.5-fold to 13 to 17 receptors/cell (KD = 260 to 300 pmol/L). That all of the Epo receptor- potentiating activity in U937CM was accounted for by EPA was shown by a similar increase in Epo receptor expression on K562 cells with recombinant EPA. The effect of U937CM on Epo receptors was reversed by culturing cells in inducer-free medium for 3 days. Medium conditioned by the 5637 cell line had no effect on Epo receptors on K562 cells. In methylcellulose culture, U937CM and Epo acted synergistically to increase erythroid differentiation of K562. Similarly, U937CM stimulated human cord blood CFU-E growth under conditions in which Epo was limiting or in excess. Increases in Epo receptor expression on K562 cells and on CFU-E in response to EPA may mediate the effects of Epo on these cells.