Immunophenotype of hematopoietic stem cells from placental/umbilical cord blood after culture

Identification and enumeration of human hematopoietic stem cells remain problematic, since in vitro and in vivo stem cell assays have different outcomes. We determined if the altered expression of adhesion molecules during stem cell expansion could be a reason for the discrepancy. CD34+CD38- and CD34+CD38+ cells from umbilical cord blood were analyzed before and after culture with thrombopoietin (TPO), FLT-3 ligand (FL) and kit ligand (KL; or stem cell factor) in different combinations: TPO + FL + KL, TPO + FL and TPO, at concentrations of 50 ng/mL each. Cells were immunophenotyped by four-color fluorescence using antibodies against CD11c, CD31, CD49e, CD61, CD62L, CD117, and HLA-DR. Low-density cord blood contained 1.4 ± 0.9 percent CD34+ cells, 2.6 ± 2.1 percent of which were CD38-negative. CD34+ cells were isolated using immuno-magnetic beads and cultured for up to 7 days. The TPO + FL + KL combination presented the best condition for maintenance of stem cells. The total cell number increased 4.3 ± 1.8-fold, but the number of viable CD34+ cells decreased by 46 ± 25 percent. On the other hand, the fraction of CD34+CD38- cells became 52.0 ± 29 percent of all CD34+ cells. The absolute number of CD34+CD38- cells was expanded on average 15 ± 12-fold when CD34+ cells were cultured with TPO + FL + KL for 7 days. The expression of CD62L, HLA-DR and CD117 was modulated after culture, particularly with TPO + FL + KL, explaining differences between the adhesion and engraftment of primary and cultured candidate stem cells. We conclude that culture of CD34+ cells with TPO + FL + KL results in a significant increase in the number of candidate stem cells with the CD34+CD38- phenotype.

Perspectivas de las células tallo en infartos del miocardio / Stem cell perspectives in myocardial infarctions

Myocardial infarction is the leading cause of congestive heart failure and death in industrializated countries. The cellular cardiomyoplasty has emerged as an alternative treatment in the regeneration of infarted myocardial tissue. In animals' models, differents cellular lines such as cardiomyocites, sheletal myoblast, embryonic stem cells and adult mesenchymal stem cells has been used, resulting in an improvement in ventricular function and decrease in amount of infarted tissue. The first three cells line have disvantages as they are allogenics and are difficult to obtain. The adult mesenchymal stem cells are autologous and can be obtained throught the aspiration of bone marrow or from peripherical circulation, prior to stimulating with cytokines (G-CSF). The implantation in humans with recent and old myocardial infarction have shown improvements similar to those shown in animal models. These findings encourage the continued investigation in the mechanism of cellular differentiation and implantation metods in infarted myocardial tissue.

El infarto del miocardio es la principal causa de falla cardiaca y muerte en países industrializados. A la fecha, la cardiomioplastia celular ha emergido como una alternativa en la regeneración de infartos miocárdicos. En modelos animales se han utilizado diferentes líneas celulares como cardiomiocitos fetales, mioblastos de músculo esquelético, células tallo embrionarias y células tallo mesenquimales del adulto, con mejoría en la función ventricular y disminución del área de tejido infartado. Las tres primeras líneas celulares tienen desventajas porque son alogénicas y difíciles de obtener. Las células tallo mesenquimales del adulto son autólogas y se pueden obtener de aspirados de médula ósea o de la circulación periférica previa estimulación con citocinas (G-CSF). La implantación de estas células en seres humanos con infartos del miocardio recientes y antiguos han mostrado mejorías similares a los reportes con modelos animales. Estos hallazgos alientan a continuar la investigación clínica y básica en busca de los mecanismos de diferenciación celular y selección de vías de implantación, en tejido miocárdico infartado.

CKbeta8-1 alters expression of cyclin E in colony forming units-granulocyte macrophage (CFU-GM) lineage from human cord blood CD34 + cells

A C6 beta-chemokine, CKbeta8-1, suppressed the colony formation of CD34 + cells of human cord blood (CB). Molecular mechanisms involved in CKbeta8-1-medicated suppression of colony formation of CD34 + cells are not known. To address this issue, the level of various G1/S cell cycle regulating proteins in CKbeta8-1-treated CD34 + cells were compared with those in untreated CD34 + cells. CKbeta8-1 did not significantly alter the expression of the G1/S cycle regulation proteins (cyclin D1, D3, and E), CDK inhibitor (p27and Rb), and other cell proliferation regulation protein (p53) in CB CD34 + cells. Here we describe an in vitro system in which CB CD34 + cells were committed to a multipotent progenitor lineage of colony forming units-granulocyte/macrophage (CFU-GM) by a simple combination of recombinant human (rh) GM-CSF and rhIL-3. In this culture system, we found that cyclin E protein appeared later and disappeared faster in the CKbeta8-1-treated cells than in the control cells during CFU-GM lineage development. These findings suggested that cyclin E may play a role in suppressing the colony formation of CFU-GM by CKbeta8-1.

Influence of genotype and explant source on indirect organogenesis by in vitro culture of leaves of Melia azedarach L

In vitro regeneration of shoots from leaf explants of the Paradise tree (Melia azedarach L.) was studied. Three different portions (proximal portion, distal portion and rachis of the leaflets) of three developmental stages (folded, young still expanding and completely expanded) of leaves of 10-15 year old plants of seven genotypes were cultured on Murashige and Skoog (1962) medium (MS) supplemented with 1 mg x l(-1) benzylaminopurine (BAP) + 0.1 mg x l(-1) kinetin (KIN) + 3 mg x l(-1) adenine sulphate (ADS). The rachis of the leaflets of the completely expanded leaves was found to be the most responsive tissue, in most of the genotypes employed. Shoot regeneration occurred in leaf explants of all the genotypes tested. The best genotype for shoot regeneration was clone 4. Rooting was induced on MS medium supplemented with 2.5 mg x l(-1) 3-indolebutyric acid, IBA, (4 days) followed by subculture on MS lacking growth regulators (26 days). Complete plants were transferred to soil.

Periodontal regeneration--fantasizing the future.

Periodontal Regenerative Surgical Procedures are, by definition, supposed to restore all three tooth-supporting tissues i.e. periodontal ligament fibres, alveolar bone and the cementum. In humans, we can now regenerate considerable amounts of alveolar bone, and periodontal ligament with the use of bone graft material and barrier membranes. The purpose of this article is to acquaint the reader with the current trends as well as some new areas along which periodontal regenerative therapy might shape up in the future. Since, some fields are still in their infancy, it can be only envisioned as to how these new fields of science might constantly evolve to redefine periodontal therapy in due time.

Neogénesis y regeneración de las células beta pancreáticas / Pancreatic beta-cell neogenesis and regeneration

OBJECTIVE: To review the literature on beta-cell neogenesis and regeneration, with special interest in substances that regulate such processes. DESIGN: Representative papers were selected through a computer MEDLINE search from 1990 to 2000. RESULTS: Several studies showed that once islets of Langerhans developed from small pancreatic ducts, this process did not continue in normal adult individuals. However, it has been published that new beta-cell formation can occur in vivo in certain experimental models through neogenesis, in which gene pax 4 is extremely important. On the other hand, substances that stimulate the regenerative process of beta-cells include glucose, several hormones and certain growth factors. CONCLUSIONS: Substances that stimulate the regenerative process commonly express this effect in elevated, non-physiologic concentrations; thus, their possible therapeutic importance is not yet clear. Nevertheless, the Reg protein, present in regenerative pancreas and implied in neogenesis process, has actual possibilities of becoming therapeutically important.

Fitoestrógenos: una nueva preocupación en la alimentación infantil / Phytoestrogens: a new preoccupation in paediatric nutrition

Los fitoestrógenos son sustancias ambientales naturales, producidas por plantas, que a pesar de su estructura química distinta de los estrógenos, actúan como tales. Estudios en adultos sugieren que tendrían efectos protectores para cánceres hormonodependientes (de próstata y mama), dislipidemias y de la mineralización ósea. Se clasifican en isoflavonas, cumestanos y lignanos, y se encuentran principalmente en legumbres y poroto de soya, brotes de poroto, forraje y granos, y en cereales de grano entero y semillas, respectivamente. Estudios recientes han demostrado que los alimentos infantiles, incluyendo algunas fórmulas lácteas, yogur y alimentos de soya, contienen cantidades considerables de fitoestrógenos. Los efectos de estos sobre la salud infantil no han sido del todo aclarados. Existen evidencias epidemiológicas y clínicas de que al actuar como estrógenos débiles prodrían determinar adelanto de los eventos puberales y telarquia en la niña y ginecomastia en el varón. Se hace una revisión del tema y se plantea la necesidad de realizar estudios destinados a aclarar los efectos de los estrógenos ambientales sobre la salud infantil

Ideal concentration of growth factors in rabbit's flexor tendon culture

Growth factors have the ability to stimulate matrix synthesis and cell proliferation in rabbit flexor tendon. Maximal stimulation effects of growth factors have a wide variation. It depends upon the different anatomic sites of the tendon segment, the kinds of growth factor, the concentration of growth factors, and the time sequence. Since proliferation was an early component of intrinsic tendon healing, we investigated the short-term dose response to four different growth factors on in vitro rabbit's tendon culture. We evaluated the effects according to the various concentrations of recombinant human insulin-like growth factor 1 (IGF), recombinant human epidermal growth factor (EGF), fibroblast growth factor (FGF), and recombinant human platelet-derived growth factor-BB (PDGF). Fetal calf serum was the most potent stimulator of cell proliferation and protein synthesis in in vitro rabbit's tendon culture. Matrix synthesis and cell proliferation were stimulated dose-dependently by IGF between the doses of 50 and 150 ng/ml. The maximum mitogenic effect of EGF was observed at the concentration of 100 ng/ml (1.3 times more than the media-only control culture). The rabbit's tendon responded significantly dose-dependently to PDGF, whereas there was no significant response to FGF.

Induction of proliferation in resting B-cells by a factor released by activated mouse spleen cells

Mouse spleen cells activated in a mixed lymphocyte reaction release a soluble factor, which induces a significant proliferative response in fresh mouse spleen cells. This proliferation inducing factor (PIF) was found to be heat stable (90 degrees C for 45 min) and also resistant to trypsin or chymotrypsin treatment. By using a sizing HPLC column, the molecular weight of PIF appears to be 25 kDa. Mouse spleen cells treated with anti-thy-1 + complement lost Con-A induced proliferative responses but responded well to PIF. B cell depleted spleen cells obtained by negative selection panning, did not respond to PIF. These results indicate that B cells proliferated in response to PIF. Polymixin-B, which blocks the B cell proliferative response to LPS, did not inhibit PIF induced proliferation.

Participaçäo de fatores de crescimento na regeneraçäo celular pós-necrose tubular aguda / Participation of growth factors in cell regeneration after acute tubular necrosis

A recuperaçäo da funçao renal após a necrose tubular aguda (NTA) depende da proliferaçäo de células e da substituiçäo de células necróticas e lesadas por células recém-formadas. Várias linhas de evidências indicam que o EGF (Epidermal Growth Factor), IGF-I (Insulin Growth Factor-I) e o HGF (Hepatocyte Growth Factor) devem participar da regeneraçäo célular pós-NTA: a) o EGF e o HGF säo capazes de desencadear potentes estímulos mitóticos em células de túbulos proximais; b) receptores para EGF e IGF-I estäo presentes em células de túbulos proximais; c) após a NTA ocorre um aumento dos receptores renais para IGF-I e EGF; d) a administraçäo de EGF, IGF-I ou HGF a ratos com NTA aumenta a velocidade de regeneraçäo celular e de recuperaçäo da funçao renal e e) o IGF-I é capaz de induzir a proliferaçäo de células renais e além disso tem efeitos no anabolismo celular e na hemodinâmica renal (aumenta o fluxo plasmático renal e a taxa de filtraçao glomerular). Tem sido proposto por vários autores o uso terapêutico de um ou mais desse fatores em pacientes com NTA. Contudo, consideramos necessária a realizaçäo de maior número de estudos para melhor entendimento dos mecanismos de açäo desses polipeptideos e avaliaçäo de seus efeitos na NTA.

Effect of embryo density and growth factors on in vitro preimplantation development of mouse embryos

Embryo development depends on maternal and embryonic factors that may regulate genetic programs in early development. Effects of growth factors on proliferation, differentiation and morphogenesis along embryogenesis have been documented. However, studies have not established the role of growth factors in the preimplantational period. The purpose of this study was to investigate the possible effects of growth factors and embryo density on mouse preimplantation development in vitro. Two-and eight-cell CF-1 embryos were cultured individually or in groups of ten in HTF medium, alone or with EGF, TGF-beta1 and IGF-I. Cleavage rate varied greatly with growth factors and increased significantly when eight-cell embryos were cultured in groups. On the other hand, when two-cell embryos were cultured in group, the cleavage rate was slower than that obtained when embryos were individually cultured. The differentiation rate increased significantly in two-cell embryos cultured in groups (p<0.05). EGF, TGF-beta1 and IGF-I increased differentiation rates significantly in two-cell embryos individually cultured for 68 hours. The combination of EGF and TGF-beta1 increased the differentiation rates significantly. The other combinations were not effective in modifying this parameter. Hatching rates increased in embryos cultured in groups (p<0.05). TGF-beta1 decreased this parameter significantly in two-or eight-cell embryos cultured in groups (p<0.05). The data described in this report suggest that preimplantational mouse embryos produce some factor or factors that enhance its development, specially the differentiation and hatching rates. However, a functional role for polypeptide growth factors during preimplantational development has to be determined.

Effect of some new insect growth regulators on metamorphosis & reproduction of Aedes aegypti.

Fourth instar larvae and pupae of Ae. aegypti were treated with four most active insect growth regulators from a new series of mixed alkyl and aryl diethers based on geraniol. Considerable reduction in fecundity and fertility of adults was obtained. Treatment of pupae or pharate adults did not affect adult emergence. Topical treatment of adult females caused great reduction in fertility and fecundity in older as compared to younger females. In addition to the effects on reproduction, adult survival was also reduced in the treated younger females.

Stimulation of intact rat liver by exogenous hepatotrophic factors with additional growth of its mass

It was previously demonstrated that it is possible to increase the size of the intact liver of rats, contray to the biological determination that ties liver size to animal size bay intraperitoneal (portal) administration of exogenous hepatotrophic factors (a solution containing glucose, amino acids, insulin, glucagon, vitamins, and electrolytes) which qualitatively mimie substances and hormones normally present in splanchnic blood (PARRA et al.). In the present investigation, we studied the possible action of fat (LipofundinR), human milk growth factors, co-factors (folic acid, vitamin B12 and zinc), and thyroid hormone (T3 on the regenerative stimulus when added to the above solution. T3 was found to be an important factor which increased liver mass when compared to the basic solution (67.54 por cento) versus 36.07 por cento). Although these values were not compared statistically due to the small number of surviving animals, they are important as guidelines for future research about new compositions of hepatotrophic solutions that will maintain or improve the level of stimulation obtained thus far, with a possible reduction in animal mortality

Regeneraçäo do fígado intacto de ratos com crescimento adicional de sua massa, estimulado por fatores hepatotróficos exógenos / Intact liver regeneration of rats with additional growth of its mass, stimulated by exogenous hepatotrophic factors

Foi anteriormente demonstrado em ratos que é possível aumentar o tamanho do fígado intacto, contrariando determinaçäo biológica que o vincula ao porte do animal, com a administraçäo intraperitoneal (portal) de fatores hepatotróficos exógenos (soluçäo de glicose, aminoácidos, insulina, glucagon, vitaminas e eletrólitos) que mimetizam, em qualidade, substâncias e hormônios normalmente existentes no sangue esplâncnico (PARRA e col.) Neste trabalho estuda-se a possível açäo, sobre o estímulo regenerativo, de gordura (Lipofundin, fatores de crescimento do leite humano, co-fatores (ácido fólico, vitamina B12 e zinco) e hormônio tiroideano (T3), quando adicionados à soluçäo anterior. Verificou-se que o T3 é fator importante, tendo aumentado a massa hepática em relaçäo à soluçäo basal (67,54// versus 36,07//). Embora tais valores näo tenham sido comparados estatísticamente pelo reduzido número de animais sobreviventes, säo todavia importantes para orientar futura pesquisa que vise buscar nova composiçäo para a soluçäo de fatores hepatotróficos que mantenha ou melhore o nível de estímulo já conseguido com possível reduçäo na mortalidade dos animais

Effect of porcine bone morphogenetic protein on healing of bone defect in the rabbit radius

Segmental long bone defects due to infection or trauma is a difficult problem to manage in patients. We studied the effect of porcine bone morphogenetic protein (pBMP) on healing of defects in the rabbit radius. Porcine BMP was separated and purified from the tibia and femur of pigs by repeated solubilization and precipitation of the protein with different concentrations of urea and GuHCl. The osteoinductive activity of pBMP was confirmed by bioassay using No. 615 mice. In rabbits, about a 15 mm length of radii were removed and 20 mg of pBMP was implanted in the defected area with fibrin sealant (FS), while only FS was implanted in controls. Union of the affected area was observed in 6 weeks in the experimental side. There was no definite evidence of bone bridging across the affected area in the controls. This suggests that pBMP has a bone forming activity in other species and the clinical use of pBMP in treating patients with segmental bone defects is promising.

Neoplastic transformation of 3T3 mouse embryo cells with c-myc- and c-Ha-ras-1 oncogenes

1. Peptide growth factors and products of some oncogenes are likely to be active in common regulatory pathways that control the cell cycle. 2. Cell transformation by DNA -mediated transfections with cloned oncogenes is an approach that can provide insight into the mechanisms of both growth factor action and cell cycle regulation. 3. This paper deals with this approach, summarizing and discussing transfection experiments of mouse c-myc- and human c-Ha-ras-1 cloned oncogenes into mouse embryo Balb-3T3 cells

Effect of leprosy sera and foetal calf serum (FCS) on the T cell number of peripheral blood of leprosy patients.

Study was conducted in 24 cases of various types of leprosy and 10 healthy controls to find out the effect of various sera on the T cell count of peripheral blood lymphocytes by sheep erythrocyte rosetting method. The percentage of T lymphocytes in lepromatous and tuberculoid cases were significantly lower compared to that in normal healthy controls. All sera except FCS had a stimulatory effect on the number of T cells. The cells incubated for 24 hours in FCS did not show any stimulatory effect on the number of T cells, however, these FCS incubated cells showed a significant elevation in the number of T cells when further incubated in sera either from leprosy cases or from healthy subjects.