Protocol for collection and separation of bone marrow mononuclear cells in Chlorocebus aethiops

Abstract: Chlorocebus aethiops is a species of non-human primate frequently used in biomedical research. Some research involves this species as an experimental model for various diseases and possible treatment with stem cells. The bone marrow is one of the main sources of these cells and provides easy access. The aim of this study was to standardize the protocol of collection and separation of bone marrow in C. aethiops. Ten animals were submitted to puncture of bone marrow with access to the iliac crest and cell separation by density gradient. The bone marrow of C. aethiops had an average of 97% viability. From the results achieved, we can conclude that C. aethiops is an excellent model to obtain and isolate mononuclear cells from bone marrow, fostering several studies in the field of cell therapy.

New strategies in hematopoietic stem cell transplantation: G-CSF-mobilized unprocessed whole blood

Transplantation of mobilized peripheral blood stem cells (PBSC) for rescue of bone marrow function after high-dose chemo-/radiotherapy is widely used in hematologic malignancies and solid tumors. Mobilization of stem cells to the peripheral blood can be achieved by cytokine treatment of the patients. The main advantage of autologous PBSC transplantation over bone marrow transplantation is the faster recovery of neutrophil and platelet counts. The threshold number of PBSC required for adequate rescue of bone marrow is thought to be about 2 x 106 CD34+ cells/kg, if the stem cells are collected by leukapheresis and subsequently cryopreserved. We show that this critical number could be further reduced to as few as 0.2 x 106 cells/kg. In 30 patients with multiple myeloma and 25 patients with bad risk lymphoma 1 liter of granulocyte colony-stimulating factor (G-CSF)-mobilized unprocessed whole blood (stored at 4oC for 1-3 days) was used for transplantation. Compared to a historical control group, a significant reduction in the duration of neutropenia, thrombocytopenia and the length of hospital stay was documented. Furthermore, the effect of stem cell support was reflected by a lower need for platelet and red cell transfusions and a reduced antibiotic use. Considering the data as a whole, a cost saving of about 50% was achieved. To date, this easy to perform method of transplantation is only feasible following high-dose therapies that are completed within 72 h, since longer storage of unprocessed blood is accompanied by a substantial loss of progenitor cell function. Ongoing investigations include attempts to prolong storage times for whole blood

Sec61alpha synthesis is enhanced during translocation of nascent chains of collagen type IV in F9 teratocarcinoma cells after retinoic acid treatment

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel called translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as being essential for translocation of polypeptide chains into the cisterns of the ER. Sec61alpha forms a protein complex with collagen and Hsp47, an ER-resident heat shock protein that binds specifically to collagen. However, it is not known whether Sec61alpha is ubiquitously produced in collagen-producing F9 teratocarcinoma cells or under heat shock treatment. Furthermore, the production and utilization of Sec61alpha may depend on the stage of cell differentiation. Cultured F9 teratocarcinoma cells are capable of differentiation in response to low concentrations of retinoic acid. This differentiation results in loss of tumorigenicity. Mouse F9 cells were grown in culture medium at 37ºC and 43ºC (heat shock treatment) treated or not with retinoic acid, and labeled in certain instances with 35S-methionine. Membrane-bound polysomes of procollagen IV were then isolated. Immunoprecipitation and Western blot analysis were performed using polyclonal antibodies against collagen IV, Hsp47 and Sec61alpha. Under retinoic acid-untreated conditions, F9 cells produced undetectable amounts of Sec61alpha. Sec61alpha, Hsp47 and type IV collagen levels were increased after retinoic acid treatment. Heat shock treatment did not alter Sec61alpha levels, suggesting that Sec61alpha production is probably not affected by heat shock. These data indicate that the enhanced production of Sec61alpha in retinoic acid-induced F9 teratocarcinoma cells parallels the increased synthesis of Hsp47 and collagen type IV.

The role of osteoblasts in regulating hematopoietic stem cell activity and tumor metastasis

Bone marrow stromal cells are critical regulators of hematopoiesis. Osteoblasts are part of the stromal cell support system in bone marrow and may be derived from a common precursor. Several studies suggested that osteoblasts regulate hematopoiesis, yet the entire mechanism is not understood. It is clear, however, that both hematopoietic precursors and osteoblasts interact for the production of osteoclasts and the activation of resorption. We observed that hematopoietic stem cells (HSCs) regulate osteoblastic secretion of various growth factors, and that osteoblasts express some soluble factors exclusively in the presence of HSCs. Osteoblasts and hematopoietic cells are closely associated with each other in the bone marrow, suggesting a reciprocal relationship between them to develop the HSC niche. One critical component regulating the niche is stromal-derived factor-1 (SDF-1) and its receptor CXCR4 which regulates stem cell homing and, as we have recently demonstrated, plays a crucial role in facilitating those tumors which metastasize to bone. Osteoblasts produce abundant amounts of SDF-1 and therefore osteoblasts play an important role in metastasis. These findings are discussed in the context of the role of osteoblasts in marrow function in health and disease.

Comparative quantification of umbilical cord blood CD34+ and CD34+ bright cells using the ProCount™-BD and ISHAGE protocols

The total number of CD34+ cells is the most relevant clinical parameter when selecting human umbilical cord blood (HUCB) for transplantation. The objective of the present study was to compare the two most commonly used CD34+ cell quantification methods (ISHAGE protocol and ProCount™ - BD) and analyze the CD34+ bright cells whose 7-amino actinomycin D (7AAD) analysis suggests are apoptotic or dead cells. Twenty-six HUCB samples obtained at the Placental Blood Program of New York Blood Center were evaluated. The absolute numbers of CD34+ cells evaluated by the ISHAGE (with exclusion of 7AAD+ cells) and ProCount™ (with exclusion of CD34+ bright cells) were determined. Using the ISHAGE protocol we found 35.6 ± 19.4 CD34+ cells/µL and with the ProCount™ method we found 36.6 ± 23.2 CD34+ cells/µL. With the ProCount™ method, CD34+ bright cell counts were 9.3 ± 8.2 cells/µL. CD34+ bright and regular cells were individually analyzed by the ISHAGE protocol. Only about 1.8% of the bright CD34+ cells are alive, whereas a small part (19.0%) is undergoing apoptosis and most of them (79.2%) are dead cells. Our study showed that the two methods produced similar results and that 7AAD is important to exclude CD34 bright cells. These results will be of value to assist in the correct counting of CD34+ cells and to choose the best HUCB unit for transplantation, i.e., the unit with the greatest number of potentially viable stem cells for the reconstitution of bone marrow. This increases the likelihood of success of the transplant and, therefore, the survival of the patient.

Inducing mutations in the mouse genome with the chemical mutagen ethylnitrosourea

When compared to other model organisms whose genome is sequenced, the number of mutations identified in the mouse appears extremely reduced and this situation seriously hampers our understanding of mammalian gene function(s). Another important consequence of this shortage is that a majority of human genetic diseases still await an animal model. To improve the situation, two strategies are currently used: the first makes use of embryonic stem cells, in which one can induce knockout mutations almost at will; the second consists of a genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes and subsequent identification of the genetic alteration(s). Several projects are now in progress making use of one or the other of these strategies. Here, we report an original effort where we mutagenized BALB/c males, with the mutagen ethylnitrosourea. Offspring of these males were screened for dominant mutations and a three-generation breeding protocol was set to recover recessive mutations. Eleven mutations were identified (one dominant and ten recessives). Three of these mutations are new alleles (Otop1mlh, Foxn1sepe and probably rodador) at loci where mutations have already been reported, while 4 are new and original alleles (carc, eqlb, frqz, and Sacc). This result indicates that the mouse genome, as expected, is far from being saturated with mutations. More mutations would certainly be discovered using more sophisticated phenotyping protocols. Seven of the 11 new mutant alleles induced in our experiment have been localized on the genetic map as a first step towards positional cloning.

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel™-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

Immune cells and oxidative stress in the endotoxin tolerance mouse model

Sepsis is a systemic inflammatory response that can lead to tissue damage and death. In order to increase our understanding of sepsis, experimental models are needed that produce relevant immune and inflammatory responses during a septic event. We describe a lipopolysaccharide tolerance mouse model to characterize the cellular and molecular alterations of immune cells during sepsis. The model presents a typical lipopolysaccharide tolerance pattern in which tolerance is related to decreased production and secretion of cytokines after a subsequent exposure to a lethal dose of lipopolysaccharide. The initial lipopolysaccharide exposure also altered the expression patterns of cytokines and was followed by an 8- and a 1.5-fold increase in the T helper 1 and 2 cell subpopulations. Behavioral data indicate a decrease in spontaneous activity and an increase in body temperature following exposure to lipopolysaccharide. In contrast, tolerant animals maintained production of reactive oxygen species and nitric oxide when terminally challenged by cecal ligation and puncture (CLP). Survival study after CLP showed protection in tolerant compared to naive animals. Spleen mass increased in tolerant animals followed by increases of B lymphocytes and subpopulation Th1 cells. An increase in the number of stem cells was found in spleen and bone marrow. We also showed that administration of spleen or bone marrow cells from tolerant to naive animals transfers the acquired resistance status. In conclusion, lipopolysaccharide tolerance is a natural reprogramming of the immune system that increases the number of immune cells, particularly T helper 1 cells, and does not reduce oxidative stress.

Cultivation and identification of colon cancer stem cell-derived spheres from the Colo205 cell line

Our group established a method to culture spheres under serum-free culture condition. However, the biological characteristics and the tumorigenicity of spheres are unknown. Here, we demonstrate that sphere cells expressed high levels of the putative colorectal cancer stem cell markers CD133 and CD44. The CD133-positive rates were 13.27 ± 5.62, 52.71 ± 16.97 and 16.47 ± 2.45% in sphere cells, regular Colo205 cells and differentiated sphere cells, respectively, while the CD44-positive rates were 62.92 ± 8.38, 79.06 ± 12.10 and 47.80 ± 2.5%, respectively, and the CD133/CD44-double-positive rates were 10.77 ± 4.96, 46.89 ± 19.17 and 12.41 ± 2.27%, respectively (P < 0.05). Cancer sphere cells formed crypt-like structures in 3-D culture. Moreover, cells from cancer spheres exhibited more tumorigenicity than regular Colo205 cells in a xenograft assay. The cancer sphere cells displayed much higher oncogenicity than regular Colo205 cells to initiate neoplasms, as assayed by H&E staining, Musashi-1 staining and electron microscopy. Our findings indicated that the sphere cells were enriched with cancer stem cells (CSCs), and exhibited more proliferation capacity, more differentiation potential and especially more tumorigenicity than regular Colo205 cells in vitro and in vivo. Further isolation and characterization of these CSCs may provide new insights for novel therapeutic targets and prognostic markers.

The role of oxytocin in cardiovascular regulation

Studies of body volume expansion have indicated that lesions of the anteroventral third ventricle and median eminence block the release of atrial natriuretic peptide (ANP) into the circulation. Detailed analysis of the lesions showed that activation of oxytocin (OT)-ergic neurons is responsible for ANP release, and it has become clear that activation of neuronal circuitry elicits OT secretion into the circulation, activating atrial OT receptors and ANP release from the heart. Subsequently, we have uncovered the entire functional OT system in the rat and the human heart. An abundance of OT has been observed in the early development of the fetal heart, and the capacity of OT to generate cardiomyocytes (CMs) has been demonstrated in various types of stem cells. OT treatment of mesenchymal stem cells stimulates paracrine factors beneficial for cardioprotection. Cardiovascular actions of OT include: i) lowering blood pressure, ii) negative inotropic and chronotropic effects, iii) parasympathetic neuromodulation, iv) vasodilatation, v) anti-inflammatory activity, vi) antioxidant activity, and vii) metabolic effects. OT actions are mediated by nitric oxide and ANP. The beneficial actions of OT may include the increase in glucose uptake by CMs and stem cells, reduction in CM hypertrophy, oxidative stress, and mitochondrial protection of several cell types. In experimentally induced myocardial infarction in rats, continuous in vivo OT delivery improves cardiac healing and cardiac work, reduces inflammation, and stimulates angiogenesis. Because OT plays anti-inflammatory and cardioprotective roles and improves vascular and metabolic functions, it demonstrates potential for therapeutic use in various pathologic conditions.

Cytokine expression patterns and mesenchymal stem cell karyotypes from the bone marrow microenvironment of patients with myelodysplastic syndromes

The purpose of this study was to explore cytokine expression patterns and cytogenetic abnormalities of mesenchymal stem cells (MSCs) from the bone marrow microenvironment of Chinese patients with myelodysplastic syndromes (MDS). Bone marrow samples were obtained from 30 cases of MDS (MDS group) and 30 healthy donors (control group). The expression pattern of cytokines was detected by customized protein array. The karyotypes of MSCs were analyzed using fluorescence in situ hybridization. Compared with the control group, leukemia inhibitory factor, stem cell factor (SCF), stromal cell-derived factor (SDF-1), bone morphogenetic protein 4, hematopoietic stem cell (HSC) stimulating factor, and transforming growth factor-β in the MDS group were significantly downregulated (P<0.05), while interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and programmed death ligand (B7-H1) were significantly upregulated (P<0.05). For chromosome abnormality analysis, the detection rate of abnormal karyotypes (+8, -8, -20, 20q-, -Y, -7, 5q-) was 30% in the MDS group and 0% in the control group. In conclusion, the up- and downregulated expression of these cytokines might play a key role in the pathogenesis of MDS. Among them, SCF and SDF-1 may play roles in the apoptosis of HSCs in MDS; and IFN-γ, TNF-α, and B7-H1 may be associated with apoptosis of bone marrow cells in MDS. In addition, the abnormal karyotypes might be actively involved in the pathogenesis of MDS. Further studies are required to determine the role of abnormal karyotypes in the occurrence and development of MDS.