Progenitor Cell Content Research Articles

A Prospective Comparison of 3 Approved Systems for Autologous Bone Marrow Concentration Demonstrated Nonequivalency in Progenitor Cell Number and Concentration

To evaluate the efficacy of 3 commercially available systems: the Harvest SmartPReP 2 BMAC, Biomet BioCUE, and Arteriocyte Magellan systems. We compared the number and concentration of progenitor cells achieved both before and after centrifugation and the percentage of progenitor cells salvaged after centrifugation. Forty patients, mean age 47 ± 18 years (range: 18-92 years, 19 male/21 female) were prospectively consented for bilateral iliac crest aspiration. The first 20 aspirations compared the Harvest and Biomet systems, and based on those results, the second 20 compared the Harvest and Arteriocyte systems. One system was randomly assigned to each iliac crest. Each system's unique marrow acquisition process and centrifugation mechanism was followed. Samples for analysis were taken both immediately before the marrow was put into the centrifugation system (after acquisition), and after centrifugation. The number of progenitor cells in each sample was estimated by counting the connective tissue progenitors (CTPs). The Harvest system achieved a significantly greater number and concentration of CTPs both before and after centrifugation when compared to the Biomet system. There was no difference in the percent yield of CTPs after centrifugation. There was no significant difference in the number and concentration of CTPs between the Harvest and Arteriocyte systems before centrifugation, but the Harvest system had a significantly greater number and concentration of CTPs after centrifugation. The Harvest system also had a significantly higher percent yield of CTPs after centrifugation compared with the Arteriocyte system. The Harvest system resulted in a greater CTP number and concentration after centrifugation when compared with the Biomet and Arteriocyte systems and may thus provide increased osteogenic and chondrogenic capacity.

D609-mediated inhibition of ATP synthesis in neural progenitor cells.

Tricyclodecan-9-yl-xanthogenate (D609) is an antioxidative molecule with antiproliferative and neuroprotective properties in a variety of cells. Previously, we have shown that D609 decreased the proliferation of neural progenitor cells. In this study, we examined the antioxidative property of D609 on neural progenitor cells isolated from the subventricular zone of the rat brain. Cellular oxidation was assessed by measuring the ATP content of the cells. Our results show that D609 decreased the ATP content of the neural progenitor cells by ∼40%, suggesting the possible inhibition of cellular metabolic activity. Cytochrome c oxidase (Cox), also known as complex IV of the electron transport chain, is a terminal enzyme involved in the oxidation of substrates resulting in the generation of energy required for the cellular activity. Therefore, regulating the activity of Cox could interfere with the generation of ATP, consequently affecting the proliferation of cells. Consistent with this hypothesis, we also observed a decrease in the Cox activity following the incubation of neural progenitor cells with D609. These results suggest that D609 could inhibit the activity of Cox and subsequent ATP synthesis in the neural progenitor cells.

Concentration and harvest of hepatic progenitor cells using continuous centrifuge system

Concentration and harvest of hepatic progenitor cells using continuous centrifuge system

Cryoprotective Effect of Disaccharides on Cord Blood Stem Cells with Minimal Use of DMSO.

Umbilical cord blood (UCB) is an extremely attractive source of stem cells for the treatment of various benign and malignant hematological and non-hematological disorders. To facilitate the preservation of these stem cells, 10% dimethylsulfoxide (DMSO) is widely used as cryoprotectant in cord blood banks. But it is found to be toxic at this concentration with the result of serious side effects in recipients after infusion of DMSO-cryopreserved cells. Evaluation of viability and functionality of cryopreserved hematopoietic stem cells is needed with either inclusion of nontoxic additives alone or with reduced DMSO concentration. We assessed the post thawing viability of UCB stem cells in the freezing medium containing disaccharides (sucrose or trehalose) alone and in combination with reduced amount i.e. 2% DMSO by trypan blue staining. The functionally active progenitor cells content of the optimized media was then evaluated and compared with 5% DMSO by a colony forming unit assay using methylcellulose based media. The freezing solution containing 0.2M trehalose with 2% DMSO came out to be superior in the evaluation of viability and generation of hematopoietic colonies of erythroid and myeloid lineage than 5% DMSO alone. While the percentage of viability was lower than 2% DMSO, as observed in the medium containing 0.2M trehalose or sucrose alone, with poor outcome of generation of myeloid lineage based colonies. Our study results suggest that trehalose (0.2M) with the inclusion of reduced concentration of DMSO(2%) can better replace 5%DMSO rather than complete removal of DMSO from the freezing medium.

Impairment of adipose tissue in Prader–Willi syndrome rescued by growth hormone treatment

Prader-Willi syndrome (PWS) results from abnormalities in the genomic imprinting process leading to hypothalamic dysfunction with an alteration of growth hormone (GH) secretion. PWS is associated with early morbid obesity and short stature which can be efficiently improved with GH treatment. Our aims were to highlight adipose tissue structural and functional impairments in children with PWS and to study the modifications of those parameters on GH treatment. Plasma samples and adipose tissue biopsies were obtained from 23 research centers in France coordinated by the reference center for PWS in Toulouse, France. Lean controls (n=33), non-syndromic obese (n=53), untreated (n=26) and GH-treated PWS (n=43) children were enrolled in the study. Adipose tissue biopsies were obtained during scheduled surgeries from 15 lean control, 7 untreated and 8 GH-treated PWS children. Children with PWS displayed higher insulin sensitivity as shown by reduced glycemia, insulinemia and HOMA-IR compared with non-syndromic obese children. In contrast, plasma inflammatory cytokines such as TNF-α, MCP-1 and IL-8 were increased in PWS. Analysis of biopsies compared with control children revealed decreased progenitor cell content in the stromal vascular fraction of adipose tissue and an impairment of lipolytic response to β-adrenergic agonist in PWS adipocytes. Interestingly, both of these alterations in PWS seem to be ameliorated on GH treatment. Herein, we report adipose tissue dysfunctions in children with PWS which may be partially restored by GH treatment.

Evaluation of peripheral blood stem cell quality in products transported by traditional courier or commercial overnight shipping services

Peripheral blood stem cell (PBSC) products have traditionally been transported from the collection center to a transplant center using validated volunteer courier-based procedures. Evolving airline service strategies and security policies have complicated this model of product transport. This study was designed to evaluate the feasibility of transporting PBSC products using commercial overnight shipping services, while maintaining product quality, compared to courier-transported products. Five PBSC products were collected from healthy volunteer donors and divided to evaluate product quality when transported either by volunteer courier or by commercial overnight shipping service. Products were evaluated on the day of collection and at 24, 48, and 72 hours postcollection for total nucleated cell (TNC) count, cell viability, progenitor cell numbers, and progenitor cell lineage growth potential (colony-forming units [CFUs]) to assess product composition and quality associated with each cohort. No delivery delays were encountered and all products were received intact. Measurements of product composition and quality demonstrated no differences in TNC count (p=0.893), cell viability (p=0.409), CD34+ progenitor cell content (p=0.509), or CFU-granulocyte-macrophage growth potential (p=0.827). We found no difference in product viability, progenitor cell content, or product potency in PBSC products transported either by volunteer courier or by commercial overnight shipping.

Combined intermittent hypobaric hypoxia and muscle electro-stimulation: a method to increase circulating progenitor cell concentration?

BackgroundOur goal was to test whether short-term intermittent hypobaric hypoxia (IHH) at a level well tolerated by healthy humans could, in combination with muscle electro-stimulation (ME), mobilize circulating progenitor cells (CPC) and increase their concentration in peripheral circulation.MethodsNine healthy male subjects were subjected, as the active group (HME), to a protocol involving IHH plus ME. IHH exposure consisted of four, three-hour sessions at a barometric pressure of 540 hPa (equivalent to an altitude of 5000 m). These sessions took place on four consecutive days. ME was applied in two separate 20-minute periods during each IHH session. Blood samples were obtained from an antecubital vein on three consecutive days immediately before the experiment, and then 24 h, 48 h, 4 days, 7 days and 14 days after the last day of hypoxic exposure. Four months later a control study was carried out involving seven of the original subjects (CG), who underwent the same protocol of blood samples but without receiving any special stimulus.ResultsIn comparison with the CG the HME group showed only a non-significant increase in the number of CPC CD34+ cells on the fourth day after the combined IHH and ME treatment.ConclusionCPC levels oscillated across the study period and provide no firm evidence to support an increased CPC count after IHH plus ME, although it is not possible to know if this slight increase observed is physiologically relevant. Further studies are required to understand CPC dynamics and the physiology and physiopathology of the hypoxic stimulus.

Inflammation Induces Hematopoietic Stem Cell/Progenitor Migration and Recruitment To Lymph Nodes In Mice and Humans

Under steady-state conditions a small fraction of hematopoietic stem and progenitor cells (HSPC) circulate in the bloodstream. Circulation of HSPC follows a circadian rhythm of migration from the bone marrow (BM) to blood and possibly, lymph. The functional purpose of circulating HSPC is unclear but it has been associated with homeostatic regulation of tissue-specific reservoir of HSPC, vasculogenesis and immunosurveillance. During infectious stress, HSPC are activated by inflammation mediators and chemokines and mobilized to circulation and extramedullary tissues. Side Population (SP) cells are stem cells with pluripotent differentiation ability found in a number of adult tissues. Here we have studied whether the lymph nodes (LN) are an obligatory site of transit/homing of HSPC in inflammation. To analyze whether there is accumulation of HSPC in inflammatory lymph nodes, we first analyzed the presence of SP cells in human LN. The patient median age was 34 (range 3-89). Patient-derived LN biopsies were diagnosed by the pathologist as having reactive lymphadenitis (RL, n=35, 50.7%) or cancer (n=34, 49.3.%). Cancer diagnoses were Hodgkin's Lymphoma (HL, n=12,17.4%), non-Hodgkin's lymphoma (NHL, n=20, 28.9%) or adenocarcinoma (AC, n=2, 2.9%) which associate with decreasing levels of inflammation. The biopsy-derived LN cells were stained with Hoechst 33342/propidium iodide and monoclonal antibodies against CD45, CD34, CD20, CD19, CD133 and ABCG2 surface markers. Flow cytometry analysis of LN specimens identified SP cells in 82.3% of RL LN samples (1.9±1.2%; range 0-39%), 50% of HL LN biopsies (0.7±0.6%; range 0-7.7%) and 20% of NHL LN biopsies (0,03±0.01%; range 0-0.23). Interestingly, SP cells were absent from AC LN specimens. The presence of LN SP cells was significantly associated with RL versus any of the analyzed cancer groups (P<0.001). The phenotype of human SP cells in all SP-positive LN specimens was CD45+/CD34-/CD19-/CD20-/CD133Low/neg. ABCG2 protein expression had a strong correlation with the presence of SP cell positive samples (c2=11.8, p=0.001). To identify whether inflammation was associated to HSPC migration to LN, we analyzed the SP cells and functional myeloid progenitor cell (CFU) content of different organs in adult mice treated with lipopolysaccharide (LPS, 3 mg/Kg) or vehicle control at 6 and 12 hours post-administration. LPS treatment resulted in migration of HSPC to LN (∼13-fold), liver (∼7-fold) and kidney (∼4 fold). Circulation of HSPC in peripheral blood increased (∼8 fold) but no HSPC was found in the thoracic duct or other organs after up to 12 hours post-infusion of LPS. These data suggest the co-existence of direct migration of HSPC to peripheral blood and lymphatic vessels. Migration of HSPC to the lymph is followed by efficient LN homing/retention of HSPC. In summary, we have found that inflammation associates with HSPC mobilization and recruitment to LN in mice and humans, strongly suggesting that local inflammatory signals are crucial in the regulation of the traffic and homing of HSPC. Disclosures:No relevant conflicts of interest to declare.

Angiotensin-(1–7) synergizes with colony-stimulating factors in hematopoietic recovery

Angiotensin (1-7) [A(1-7)] is a bioactive peptide of the renin angiotensin system that stimulates the number of bone marrow progenitors and hematopoietic recovery after myelosuppression. We evaluated the combination of A(1-7) with colony-stimulating factors, Neupogen and Epogen, on bone marrow progenitors and the recovery of circulating formed elements following chemotherapy. Mice were injected with gemcitabine followed 2 days later with A(1-7). Circulating blood cells and bone marrow progenitors were measured over time. Combination of A(1-7) with Neupogen (the latter given only 3 days starting at the white blood cell nadir) decreased the amount of Neupogen needed for optimal recovery by 10-fold. The progenitors measured include CFU-GEMM, CFU-GM, CFU-Meg and BFU-E. A(1-7) increased recovery of all progenitors when given alone or in combination with Neupogen above that with Neupogen alone. Combination of A(1-7) with Epogen slightly increased (not significantly) red blood cell concentrations above those achieved by Epogen alone. However, in this model, A(1-7) or A(1-7) in combination with Epogen increased all erythroid progenitors with the largest effect on early erythroid progenitors (immature BFU-E). Neupogen and Epogen acted synergistically with A(1-7) to increase the concentration of myeloid, megakaryocytic and erythroid progenitor cells in the bone marrow following chemotherapy suggesting that A(1-7)'s multilineage effect on early progenitors in the marrow facilitates proliferation in response to lineage-specific growth factors.

Mononuclear cells from a rare blood donor, after freezing under good manufacturing practice conditions, generate red blood cells that recapitulate the rare blood phenotype.

Cultured red blood cells (cRBCs) from cord blood (CB) have been proposed as transfusion products. Whether buffy coats discarded from blood donations (adult blood [AB]) may be used to generate cRBCs for transfusion has not been investigated. Erythroid progenitor cell content and numbers and blood group antigen profiles of erythroblasts (ERYs) and cRBCs generated in human erythroid massive amplification (HEMA) culture by CB (n = 7) and AB (n = 33, three females, three males, one AB with rare blood antigens cryopreserved using CB protocols) were compared. Variability was observed both in progenitor cell content (twofold) and number of ERYs generated (1 log) by CB and AB in HEMA. The average progenitor cell contents of the subset of AB and CB analyzed were similar. AB generated numbers of ERYs three times lower (p < 0.01) than CB in HEMA containing fetal bovine serum but similar to CB in HEMA containing human proteins. Female AB contained two times fewer (p < 0.05) erythroid progenitor cells but generated numbers of ERYs similar to those generated by male AB. Cryopreserved AB with a rare blood group phenotype and shipped to another laboratory generated great numbers of ERYs, 90% of which matured into cRBCs. Blood group antigen expression was consistent with the donor genotype for ERYs generated both by CB and AB but concordant with that of native RBCs only for cells derived from AB. Buffy coats from regular donors, including a donor with rare phenotypes stored under conditions established for CB, are not inferior to CB for the generation of cRBCs.

The level of circulating endothelial progenitor cells may be associated with the occurrence and recurrence of chronic subdural hematoma

OBJECTIVES:The onset of chronic subdural hematoma may be associated with direct or indirect minor injuries to the head or a poorly repaired vascular injury. Endothelial progenitor cells happen to be one of the key factors involved in hemostasis and vascular repair. This study was designed to observe the levels of endothelial progenitor cells, white blood cells, platelets, and other indicators in the peripheral blood of patients diagnosed with chronic subdural hematoma to determine the possible relationship between the endothelial progenitor cells and the occurrence, development, and outcomes of chronic subdural hematoma.METHOD:We enrolled 30 patients with diagnosed chronic subdural hematoma by computer tomography scanning and operating procedure at Tianjin Medical University General Hospital from July 2009 to July 2011. Meanwhile, we collected 30 cases of peripheral blood samples from healthy volunteers over the age of 50. Approximately 2 ml of blood was taken from veins of the elbow to test the peripheral blood routine and coagulation function. The content of endothelial progenitor cells in peripheral blood mononuclear cells was determined by flow cytometry.RESULTS:The level of endothelial progenitor cells in peripheral blood was significantly lower in preoperational patients with chronic subdural hematomas than in controls. There were no significant differences between the two groups regarding the blood routine and coagulation function. However, the levels of circulating endothelial progenitor cells were significantly different between the recurrent group and the non-recurrent group.CONCLUSIONS:The level of circulating endothelial progenitor cells in chronic subdural hematoma patients was significantly lower than the level in healthy controls. Meanwhile, the level of endothelial progenitor cells in recurrent patients was significantly lower than the level in patients without recurrence. Endothelial progenitor cells may be related to the occurrence and recurrence of chronic subdural hematoma.

Benefits of small volume and small syringe for bone marrow aspirations of mesenchymal stem cells

Aspirating bone marrow from the iliac crest using small volumes of 1-4 ml with a 10-ml syringe has been historically proposed for harvesting adult mesenchymal stem cells and described as a standard technique to avoid blood dilution. The disadvantage of repeated small aspirations is that there is a significantly increased time to harvest the bone marrow. However, it is not known if a large volume syringe can improve the rate of bone marrow aspiration without increasing blood dilution, thus reducing the quality of the aspirate. We compared the concentrations of mesenchymal stem cells obtained under normal conditions with two different size syringes. Thirty adults (16 men and 14 women with a mean age of 49 ± 14 years) underwent surgery with aspiration of bone marrow from their iliac crest. Bilateral aspirates were obtained from the iliac crest of the same patients with a 10-ml syringe and a 50-ml syringe. Cell analysis determined the frequencies of mesenchymal stem cells (as determined by the number of colonies) from each size of syringe. The cell count, progenitor cell concentration (colonies/ml marrow) and progenitor cell frequency (per million nucleated cells) were calculated. All bone marrow aspirates were harvested by the same surgeon. Aspirates of bone marrow demonstrated greater concentrations of mesenchymal stem cells with a 10-ml syringe compared with matched controls using a 50-ml syringe. Progenitor cell concentrations were on average 300 % higher using a 10-ml syringe than matched controls using a 50-ml syringe (p < 0.01). In normal human donors, bone marrow aspiration from 30 patients demonstrated a reduced mesenchymal stem cell number in aspirates obtained using a larger volume syringe (50 ml) as compared with a smaller volume syringe (10 ml).

Quantitative Assessment of the Yield of Osteoblastic Connective Tissue Progenitors in Bone Marrow Aspirate from the Iliac Crest, Tibia, and Calcaneus

It is well known that bone marrow aspirate from the iliac crest contains osteoblastic connective tissue progenitor cells. Alternative harvest sites in foot and ankle surgery include the distal aspect of the tibia and the calcaneus. To our knowledge, no previous studies have characterized the quality of bone marrow aspirate obtained from these alternative sites and compared the results with those of aspirate from the iliac crest. The goal of this study was to determine which anatomic location yields the highest number of osteoblastic progenitor cells. Forty patients were prospectively enrolled in the study, and separate bone marrow aspirate samples were harvested from the ipsilateral anterior iliac crest, distal tibial metaphysis, and calcaneal body. The aspirate was centrifuged to obtain a concentrate of nucleated cells, which were plated and grown in cell culture. Colonies that stained positive for alkaline phosphatase were counted to estimate the number of osteoblastic progenitor cells in the initial sample. The anatomic locations were compared. Clinical parameters (including sex, age, tobacco use, body mass index, and diabetes) were assessed as possible predictors of osteoblastic progenitor cell yield. Osteoblastic progenitor cells were found at each anatomic location. Bone marrow aspirate collected from the iliac crest had a higher mean concentration of osteoblastic progenitor cells compared with the distal aspect of the tibia or the calcaneus (p &lt; 0.0001). There was no significant difference in concentration between the tibia and the calcaneus (p = 0.063). Age, sex, tobacco use, and diabetes were not predictive of osteoblastic progenitor cell yield. Osteoblastic progenitor cells are available in the iliac crest, proximal aspect of the tibia, and calcaneus. However, the iliac crest provided the highest yield of osteoblastic progenitor cells. The study demonstrated that osteogenic progenitor cells are available in bone marrow aspirate harvested from the tibia or calcaneus as well as the iliac crest. All three sites are easily accessed, with a low risk of adverse events. However, larger volumes of aspirate may be needed from the tibia or calcaneus to approach the yield of cells from the iliac crest.

Identification of the chemokine CCL28 as a growth and survival factor for human hematopoietic stem and progenitor cells

In an attempt to discover novel growth factors for hematopoietic stem and progenitor cells (HSPCs), we have assessed cytokine responses of cord blood (CB)-derived CD34(+) cells in a high-content growth factor screen. We identify the immunoregulatory chemokine (C-C motif) ligand 28 (CCL28) as a novel growth factor that directly stimulates proliferation of primitive hematopoietic cells from different ontogenetic origins. CCL28 enhances the functional progenitor cell content of cultured cells by stimulating cell cycling and induces gene expression changes associated with survival. Importantly, addition of CCL28 to cultures of purified putative hematopoietic stem cells (HSCs) significantly increases the ability of the cells to long-term repopulate immunodeficient mice compared with equivalent input numbers of fresh cells. Together, our findings identify CCL28 as a potent growth-promoting factor with the ability to support the in vitro and in vivo functional properties of cultured human hematopoietic cells.

Treatment of erosive osteoarthritis with peroxisome proliferator-activated receptor alpha agonist fenofibrate: a pilot study

Hand osteoarthritis (HOA) is a common condition associated with high disease burden and frequently accompanied by comorbidities including dyslipidemia, atherosclerosis and obesity. The most debilitating HOA phenotype is erosive HOA (EHOA), characterized by synovial inflammation, formation of erosions, and substantial decline in hand function. Currently, there is no proven symptomatic treatment for the EHOA. Due to their broad spectrum effects directed on lipid metabolism, inflammation and pain, the agonists of peroxisome proliferator-activated receptor alpha or fibrates are a candidate class of drugs for the treatment of EHOA. In this study, we assessed the influence of fenofibrate treatment on clinical efficacy parameters, in vivo cytokine and adipokine production and concentrations of endothelial progenitor cells (EPC) in patients with EHOA. Fourteen patients received treatment with 145mg of fenofibrate/day for 12weeks. Fenofibrate treatment was associated with significant decreases in pain score, tender joint count, duration of morning stiffness, disease activity score, Cochin index, and ESR. Eight (57.14%) patients developed Outcome Measures in Rheumatology Clinical Trials-Osteoarthritis Research Society response at the end of treatment. Paracetamol consumption did not change during the treatment course. There was a significant reduction in triglyceride levels. No changes were detected in serum pro-inflammatory cytokine and adipokine concentrations while circulating IL-10 levels significantly decreased. There were no differences in circulating EPC numbers before and after the treatment. Fenofibrate was well tolerated, no patient experienced disease flare during the treatment. In conclusion, in EHOA patients, fenofibrate is associated with pleiotropic effects on pain, inflammation, and lipid profile. Larger, controlled studies are needed to confirm these results.

Molecular and functional characterization of human bone marrow adipocytes

Adipocytes are a cell population largely located in the human bone marrow cavity. In this specific microenvironment where adipocytes can interact with a variety of different cells, the role of fat is mainly unknown. To our knowledge, this report is the first to characterize mature adipocytes isolated from human bone marrow (BM-A) molecularly and functionally to better understand their roles into the hematopoietic microenvironment. Healthy BM-A were isolated after collagenase digestion and filtration. We studied the morphology of BM-A, their gene expression and immunophenotypic profile and their functional ability in the hematopoietic microenvironment, comparing them with adipocytes derived from adipose tissue (AT-A). BM-A showed a unilocular lipid morphology similar to AT-A and did not lose their morphology in culture; they showed a comparable pattern of stem cell-surface antigens to AT-A. In line with these observations, molecular data showed that BM-A expressed some embryonic stem cells genes, such as Oct4, KLf4, c-myc, Gata4, Tbx1, and Sox17, whereas they did not express the stem cell markers Sox2 and Nanog. Moreover, BM-A had long telomeres that were similar to bone marrow mesenchymal stem cells. Notably, BM-A supported the survival and differentiation of hematopoietic stem cells in long-term cultures. These results showed that BM-A are stromal cells with a gene expression pattern that distinguished them from AT-A. BM-A showed stem cell properties through their hematopoietic supporting function, which was certainly linked to their role in the maintenance of the bone marrow microenvironment. Depending on specific demands, BM-A may acquire different functions based on their local environment.

Lymphoid Progenitor Cells from Childhood Acute Lymphoblastic Leukemia Are Functionally Deficient and Express High Levels of the Transcriptional Repressor Gfi-1

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. Substantial progress on understanding the cell hierarchy within ALL bone marrow (BM) has been recorded in the last few years, suggesting that both primitive cell fractions and committed lymphoid blasts with immature stem cell-like properties contain leukemia-initiating cells. Nevertheless, the biology of the early progenitors that initiate the lymphoid program remains elusive. The aim of the present study was to investigate the ability of lymphoid progenitors from B-cell precursor ALL BM to proliferate and undergo multilineage differentiation. By phenotype analyses, in vitro proliferation assays, and controlled culture systems, the lymphoid differentiation potentials were evaluated in BM primitive populations from B-cell precursor ALL pediatric patients. When compared to their normal counterparts, functional stem and progenitor cell contents were substantially reduced in ALL BM. Moreover, neither B nor NK or dendritic lymphoid-cell populations developed recurrently from highly purified ALL-lymphoid progenitors, and their proliferation and cell cycle status revealed limited proliferative capacity. Interestingly, a number of quiescence-associated transcription factors were elevated, including the transcriptional repressor Gfi-1, which was highly expressed in primitive CD34+ cells. Together, our findings reveal major functional defects in the primitive hematopoietic component of ALL BM. A possible contribution of high levels of Gfi-1 expression in the regulation of the stem/progenitor cell biology is suggested.

Impact of &amp;lt;i&amp;gt;Celosia cristata&amp;lt;/i&amp;gt; Extract on Adipogenesis of Native Human CD34&amp;lt;sup&amp;gt;+&amp;lt;/sup&amp;gt;/CD31&amp;lt;sup&amp;gt;-&amp;lt;/sup&amp;gt; Cells

Background: The adipose tissue mainly consists of adipocytes but also contains non-adipose cells. Among them, progenitor cells represent a local pool of immature cells that, in vitro, can undergo various lineage differentiation processes. These cells are thought to contribute to normal homeostasis of the adipose tissue through adipogenesis but also to the growth of the adipose tissue under chronic energy overload. The aim of the present study is to evaluate in vitro the capacity of a Celosia cristata extract to impact the adipogenic potential of native human adipose tissue progenitor cells, i.e. commitment and differentiation towards adipogenic lineage. Methods: Native adipose tissue progenitor cells were isolated by immunoselection/depletion approaches from human subcutaneous adipose tissues. Two distinct cell culture conditions were used to assess the effect of Celosia cristata extract on commitment and differenciation of progenitor cells. Cells were cultured either in differentiation medium for 10 days in the presence/absence of Celosia cristata extracts to study the impact on differentiation or first cultured in a commitment-inducing medium, with or without Celosia cristata extract, for 48 h and then cultured 10 days in differentiation medium to assess the impact on commitment. In both experimental series, the fate of progenitor cells was studied by quantification of lipids and by determining the expression of key genes involved in adipogenesis. Results: Data show that Celosia cristata extract reduces lipid content of progenitor cells undergoing differentiation. This reduction correlates with a reduced expression of C/EBPα. When progenitor cells are placed in commitment-inducing conditions, Celosia cristata extract induces a more potent reduction of lipid content. This reduction correlates with a decrease in the expression levels of master genes involved in adipogenesis: the genes of transcription factors PPARγ2 and C/EBPα as well as marker genes coding for LPL and GPDH. Conclusions: Celosia cristata extract decreases adipogenesis. The effect of the extract is stronger when studying commitment and differentiation than differentiation alone; it suggests that the extract impact the commitment of human adipose tissue progenitor cells.

Analysis of Cord Blood Unit (CBU) Segment for CD34+ Cell Viability and Hematopoietic Progenitor Cell Content Correlates with the Post-Thaw CBU After Albumin-Dextran Reconstitution.

AbstractAbstract 3023 Background:The segment attached to the freezing bag is considered an important source of cells for testing the quality of a cryopreserved CBU prior to release for transplantation. CB banks test cells from the segment for CD34+ cell count, viability and colony forming units (CFU) as surrogates of potency of the frozen products. The NCBP reported results of 384 segments of CBU processed with the AutoExpress (AXP) system (Albano et al, ASH 2011). Average segment CD34+ viability was 96% (SD:+/− 3.3%) and correlation with the CBU pre-cryopreservation viable CD34+ counts (vCD34; R2:0.9) and CFU (R2:0.6) were excellent. Segment vCD34+ and CFU also correlated highly with each other (R2:0.69). As of 08/2012, 1056 segments from NCBP AXP CBU have been evaluated with average CD34+ cell viability of 96% (SD:+/− 3.1). However, how the segment results compare to those obtained from the CBU at the transplant center is not established. Methods:To evaluate whether the segment could predict the post-thaw CBU vCD34+ counts, viability and CFU at MSKCC, we compared the post-thaw results of 37 NCBP CBU, AXP-processed and stored in BioArchive freezers, shipped, and thawed at MSKCC, with the information from their respective segments tested at NCBP prior to CBU release and the pre-cryopreservation data. Segment CD34+ counts and viability were evaluated by flow cytometry and 7-AAD exclusion using a single platform and the ISHAGE gating strategy. Segment CFU were evaluated using the NCBP CFU high resolution digital imaging technology (Albano et al, ASH 2008). The segment viable cells/ul were used to estimate the total vCD34 and CFU for the respective CBU. At MSKCC, CBU underwent thaw and albumin reconstitution with 8-fold dilution (10% Dextran 40; 25% albumin) as previously reported (Barker et al, BBMT 2009;15(12):1596–602). Duplicate samples were evaluated by flow cytometry within two hours. Four color flow cytometry using a dual platform was performed to measure CD45+/CD34+/CD3+ cell counts; CD34+ cell viability was assessed using a modified ISHAGE strategy (Scaradavou et al, BBMT 2010;16(4):560–8). CFU assays were performed using 1×105cells plated in duplicate and growth was evaluated at 14 days. All CBU were part of double unit grafts. Results:Consistent with prior NCBP data, segment vCD34+ cell counts correlated well with segment CFU (R2:0.89, p< 0.01; N=21). Additionally, high correlation of vCD34+ cell counts and CFU were seen between the pre-cryopreservation CBU and the segment (p<0.01, Table).Importantly, despite the differences in testing laboratories and gating strategies, the number of vCD34+ cells in the CBU post-thaw correlated with the pre-cryopreservation vCD34+ counts, as well as those from the segment (Table). Average decrease in post-thaw CBU CD34+ cell viability compared to that of the segment was 1.4% (SD:+/− 3.7%, N: 37). Although statistically significant (p: 0.029), this difference was not clinically relevant: the range of change was -10% to +4.8% and the lowest CBU CD34+ viability was 86% (Figure). Post-thaw CBU total CFU and CFUGM did not correlate with pre-cryopreservation values (Table), probably reflecting high inter-laboratory assay variability. Segment CFU and CFUGM correlation with the post-thaw values was significant although the R2was weak. The ratio of post-thaw to segment vCD34 cells (median: 1.3; SD:+/− 0.47) indicated that the segment calculation may underestimate CBU cell content. The median ratio of post-thaw to segment colony-forming cells was 0.54 for total CFU (SD:+/− 0.63) and 0.5 for CFUGM (SD:+/− 0.96). The lower CFU than vCD34 ratio may be explained, in part, by the fact that 7-AAD detects dead cells but not apoptotic; apoptotic cells are counted as alive by flow cytometry but do not have functionality and do not generate CFU in culture. Conclusions:Our results indicate that testing of CBU segments can measure accurately the potency of the frozen CB products. Moreover, the results demonstrate that CBU quality can be maintained following albumin-dextran reconstitution. These findings reflect the cryopreservation procedures, freezing bags and reconstitution method described; whether they can be applied to other CB banks or transplant center laboratories requires further investigation. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Anemia, heart failure and exercise training

We read with interest the recent article by Wang et al. in which theauthors evaluated in anemic patients with heart failure whether exer-tionalperiodicbreathing influences rheological/hemodynamicfunctions[1]. For this purpose, the authors performed an incremental exercisetestusing a bicycle ergometer. In this regard, it has been generally assumedby some researchers, exercise physiologists and scientists that the incre-ment in blood hemoglobin (Hb) content is among the main hematolog-ical adaptations to endurance exercise training. This misconception isbased in the data published in diffe rent studies carried out severalyearsago [2].Inarecentmanuscript byBakeretal. [3],theauthorsdem-onstrated that endurance exercise training increases hematopoiesis,enhancing skeletal muscle hematopoietic cytokine expression, and mo-bilizing medullary hematopoietic stem and progenitor cell content. Themainconclusionofthisstudywasthatendurancetrainingshouldbecon-sidered as a strategy for blood donor preconditioning. The authors alsosuggestthatitwouldbeusefulasaphysiologicaladjuvantforautologousbonemarrowtransplantationorasa first-steptherapyforsomeformsofanemia. In our opinion, and in accordance with the results showed byWangandcollaborators,theseassumptionsarequestionableandagainstthe vast majority of experimental evidence.For instance, in 2000, Bojadjiev and Taralov found that continuous(formorethanoneyear)high-intensitysportstrainingcausedadecreasein blood Hb concentration in pubescent boys and girls [4].Twoyears