Adult hematopoietic progenitors are multipotent in chimeric mice

Embryonic stem cells (ESCs) and adult somatic cells, induced to pluripotency (iPSCs), can differentiate into multiple cell lineages. We previously reported that adult mammalian bone marrow contains a sub-population of CD34+ cells that express genes of ESCs and genes required to generate iPSCs. They also express lineage genes of the three embryonic germ layers. Are these CD34+ cells multipotent? Here, CD34+ bone marrow stem cells from adult male ROSA mice, which carry two markers: the β-galactosidase gene and the male Y chromosome, were transplanted into blastocysts of wildtype mice. Each female ROSA chimera generated had a distinct pattern of male-derived organs expressing β-galactosidase; e.g., ectodermal brain, dorsal root ganglia and skin; mesodermal heart, bone and bone marrow; and endodermal pancreas, intestine, and liver. Thus, adult mammals carry cells that appear to exhibit a developmental potential reminiscent of ESCs and iPSCs suggesting they could be used for cell replacement therapy.     

Adenoviral gene transfer to committed hematopoietic progenitors

High-efficiency gene transfer into ex vivo expanded human hematopoietic progenitors and precursor cells by adenovirus vectorsFrey, B.M. et al. (1998)Blood 91, 2781–2792     

Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells

In this study, we have characterized the early steps of hematopoiesis during embryonic stem cell differentiation. The immunophenotype of hematopoietic progenitor cells derived from murine embryonic stem cells was determined using a panel of monoclonal antibodies specific for hematopoietic differentiation antigens. Surprisingly, the CD41 antigen (alphaIIb integrin, platelet GPIIb), essentially considered to be restricted to megakaryocytes, was found on a large proportion of cells within embryoid bodies although very few megakaryocytes were detected. In clonogenic assays, more than 80% of all progenitors (megakaryocytic, granulo-macrophagic, erythroid and pluripotent) derived from embryoid bodies expressed the CD41 antigen. CD41 was the most reliable marker of early steps of hematopoiesis. However, CD41 remained a differentiation marker because some CD41(-) cells from embryoid bodies converted to CD41(+) hematopoietic progenitors, whereas the inverse switch was not observed. Immunoprecipitation and western blot analysis confirmed that CD41 was present in cells from embryoid bodies associated with CD61 (beta3 integrin, platelet GPIIIa) in a complex. Analysis of CD41 expression during ontogeny revealed that most yolk sac and aorta-gonad-mesonephros hematopoietic progenitor cells were also CD41(+), whereas only a minority of bone marrow and fetal liver hematopoietic progenitors expressed this antigen. Differences in CD34 expression were also observed: hematopoietic progenitor cells from embryoid bodies, yolk sac and aorta-gonad-mesonephros displayed variable levels of CD34, whereas more than 90% of fetal liver and bone marrow progenitor cells were CD34(+). Thus, these results demonstrate that expression of CD41 is associated with early stages of hematopoiesis and is highly regulated during hematopoietic development. Further studies concerning the adhesive properties of hematopoietic cells are required to assess the biological significance of these developmental changes.     

Roles of Bim in apoptosis of normal and Bcr-Abl-expressing hematopoietic progenitors

Bcr-Abl kinase is known to reverse apoptosis of cytokine-dependent cells due to cytokine deprivation, although it has been controversial whether chronic myeloid leukemia (CML) progenitors have the potential to survive under conditions in which there are limited amounts of cytokines. Here we demonstrate that early hematopoietic progenitors (Sca-1(+) c-Kit(+) Lin(-)) isolated from normal mice rapidly undergo apoptosis in the absence of cytokines. In these cells, the expression of Bim, a proapoptotic relative of Bcl-2 which plays a key role in the cytokine-mediated survival system, is induced. In contrast, those cells isolated from our previously established CML model mice resist apoptosis in cytokine-free medium without the induction of Bim expression, and these effects are reversed by the Abl-specific kinase inhibitor imatinib mesylate. In addition, the expression levels of Bim are uniformly low in cell lines established from patients in the blast crisis phase of CML, and imatinib induced Bim in these cells. Moreover, small interfering RNA that reduces the expression level of Bim effectively rescues CML cells from apoptosis caused by imatinib. These findings suggest that Bim plays an important role in the apoptosis of early hematopoietic progenitors and that Bcr-Abl supports cell survival in part through downregulation of this cell death activator.     

Stimulation of retroviral vector infection of murine hematopoietic progenitors

Conditioned media (CM) from a cloned murine marrow-derived stromal cell line, AC6.21 (ALC), was shown to stimulate retroviral vector infection of hematopoietic progenitors in culture. Inclusion of ALC CM during cocultivation of normal murine bone marrow (BM) with vector-producing fibroblasts improved infection efficiency of day 13 spleen colony-forming cells (CFU-s) from 63% (15 provirus-positive spleen colonies/24 total), without added growth factor, to 90% (36 provirus-positive colonies/40 total). In addition, stimulation of BM cells with ALC CM during cocultivation improved retroviral infection of stem cells capable of repopulating the hematopoietic system of irradiated recipient animals. Because ALC CM was found to have 50 to 100 U/ml of IL-6 activity, purified recombinant human IL-6 was tested for an effect in this system. Stimulation with IL-6 alone increased retroviral infection efficiency of CFU-s from 15% (17 colonies provirus-positive/111 total analyzed) without added growth factor to 66% (97 provirus-positive colonies/148 total analyzed). These experiments support and extend previous studies which have demonstrated the necessity for growth factor stimulation in optimizing retroviral vector transduction of hematopoietic precursors.     

Oxidative stress in athletes during extreme endurance exercise

Despite the many known health benefits of exercise, there is a body of evidence suggesting that endurance exercise is associated with oxidative stress. To determine whether extreme endurance exercise induces lipid peroxidation, 11 athletes (3 females, 8 males) were studied during a 50 km ultramarathon (trial 1) and during a sedentary protocol (trial 2) 1 month later. The evening before each trial, with dinner, subjects consumed 75 mg each d(3)-RRR and d(6)-all rac-alpha-tocopheryl acetates. Blood was obtained at baseline, 30 min pre-race, mid-race, post-race, 1 h post-race, 24 h post-race, and at corresponding times during trial 2. All 11 subjects completed the race; average run time was 391 +/- 23 min. Plasma F(2)-isoprostanes increased from 75 +/- 7 pg/ml at pre-race to 131 +/- 17 (p <.02) at post-race, then returned to baseline at 24 h post-race; F(2)-isoprostanes were unchanged during trial 2. Deuterated alpha-tocopherol disappearance rates were faster (2.8 x 10(-4) +/- 0.2 x 10(-4)) during the race compared to the sedentary trial (2.3 x 10(-4) +/- 0.2 x 10(-4); p <.03). These data suggest that extreme endurance exercise results in the generation of lipid peroxidation with a concomitant increase in vitamin E disappearance.     

Production of human hematopoietic progenitors in a clinical-scale stirred suspension bioreactor

Ex vivo expanded primitive hematopoietic cells can be utilized in bone marrow transplantation therapies to treat patients suffering from various cancers and hematopoietic malignancies. A high initial cell density (10⁶ cells/mL) and the supplement of soluble factors secreted by stromal feeders in combination with growth-promoting (interleukin-3 and stem cell factor) and growth-inhibiting (macrophage-inflammatory protein-1) cytokines resulted in high, long-term expansions (17-fold over a 14-day culture period) of human hematopoietic progenitors in a stirred suspension bioreactor. This study demonstrated that a transplantable dosage of human hematopoietic progenitor cells (8.1 ± 1.3 × 10⁶ colony forming unit-granulocyte/macrophage) can be generated from approximately 10 mL of bone marrow aspirate in a 14-day culture using a 250 mL suspension bioreactor system. © Rapid Science Ltd. 1998     

Constitutively active beta-catenin promotes expansion of multipotent hematopoietic progenitors in culture

This study was designed to investigate one component of the Wnt/beta-catenin signaling pathway that has been implicated in stem cell self-renewal. Retroviral-mediated introduction of stable beta-catenin to primitive murine bone marrow cells allowed the expansion of multipotential c-Kit(low)Sca-1(low/-)CD19(-) CD11b/Mac-1(-)Flk-2(-)CD43(+)AA4.1(+)NK1.1(-)CD3(-)CD11c(-)Gr-1(-)CD45R/B220(+) cells in the presence of stromal cells and cytokines. They generated myeloid, T, and B lineage lymphoid cells in culture, but had no T lymphopoietic potential when transplanted. Stem cell factor and IL-6 were found to be minimal requirements for long-term, stromal-free propagation, and a beta-catenin-transduced cell line was maintained for 5 mo with these defined conditions. Although multipotential and responsive to many normal stimuli in culture, it was unable to engraft several types of irradiated recipients. These findings support previous studies that have implicated the canonical Wnt pathway signaling in regulation of multipotent progenitors. In addition, we demonstrate how it may be experimentally manipulated to generate valuable cell lines.     

Effects of dolichol and dolichyl phosphate on in vitro differentiation of hematopoietic progenitors

The exogenous addition of dolichyl phosphate (Dol-P), an active form of dolichol (Dol) that carries oligosaccharide chains for protein-N-glycosylation, significantly enhanced colony formation of mouse bone marrow hematopoietic progenitors (CFU-e, BFU-e, and CFU-gm) was stimulated by erythropoietin (Epo) and colony-stimulating factor (CSF), but Dol enhanced colony formation of CFU-e only. The effects of Dol or Dol-P on these hematopoietic progenitors were fully dependent on stimulation by Epo or CSF. Other mevalonate-metabolites, such as cholesterol, coenzyme Q10, and isopentenyladenine, had no effect on hematopoietic progenitors. These studies suggest that exogenous Dol-P enhances the frequency of differentiation of hematopoietic progenitors stimulated by Epo or CSF, and there may be a diversity in cellular response of these progenitors to Dol.     

Umbilical cord blood stem cells can expand hematopoietic and neuroglial progenitors in vitro

The ability of hematopoietic tissue-derived adult stem cells to transdifferentiate into neural progenitor cells offers an interesting alternative to central nervous system (CNS)- or embryonic-derived stem cells as a viable source for cellular therapies applied to brain regeneration. Umbilical cord blood (CB) due to its primitive nature and it unproblematic collection appears as a promising candidate for multipotent stem cell harvest. We developed a negative immunomagnetic selection method that depletes CB from hematopoietic lineage marker-expressing cells, hence isolating a discrete lineage negative (LinNeg) stem cell population (0.1% of CB mononucleated cell [MCN] population). In liquid culture supplemented with thrombopoietin, flt-3 ligand, and c-kit ligand (TPOFLK), CB LinNeg stem cells could expand primitive nonadherent hematopoietic progenitors (up to 47-fold) and simultaneously produce slow-dividing adherent cells with neuroglial progenitor cell morphology over 8 weeks. Laser scanning confocal microscopy analysis identified these adherent cells to express glial fibrillary acidic protein (GFAP). Gene expression analysis showed upregulation of primitive neuroglial progenitor cell markers including, GFAP, nestin, musashi-1, and necdin. ELISA quantification of liquid culture supernatant revealed the in vitro release of transforming growth factor beta-1 (TGFbeta1), glial cell line-derived neurotrophic factor (GDNF) suggesting their contribution to CB LinNeg stem cell transdifferentiation into neuroglial progenitors. Our study supports that a single CB specimen can be pre-expanded in TPOFLK to produce both primitive hematopoietic and neuropoietic progenitors, hence widening CB clinical potential for cellular therapies.     

Dynamics of cardiac performance in athletes during isometric exercise

The main structural-functional indices of cardiac performance during isometric exercise were recorded in athletes and non-athletes. The physiological shifts in response to exercise were less obvious in the athletes than in the non-athletes. This was mainly determined by the structural features of the “athlete’s heart.” The dependence of a series of physiological indices on the heart rate was revealed: as cardiac rhythm increased in response to a submaximal isometric exercise, the systolic and, to a lesser degree, diastolic arterial pressure grew and the stroke volume of blood decreased.     

Effects of IL-1 on hematopoietic progenitors after myelosuppressive chemoradiotherapy

Recently it has been recognized that IL-1 plays an important role in hematopoietic regulation. Administration of 5-fluorouracil (5-FU) to mice causes prolonged neutropenia. rHIL-1 injected to mice after 5-FU, accelerated the recovery of hematopoietic progenitors and blood neutrophils. The combination of rhIL-1 and rhG-CSF reduced the neutropenic period significantly. Sublethal irradiation of mice induced profound neutropenia for 3 weeks which was associated with 80% mortality. Administration of rhIL-1 20 hours prior to or 2 hours post irradiation resulted in a significantly improved survival and rapid recovery of the neutrophil count. IL-1 administered alone or in combination with other colony stimulating factors to spontaneous breast tumor bearing mice following 5-FU therapy resulted in a rapid recovery of neutrophils, improved survival, and markedly reduced the tumor mass. Experiments in primates demonstrated that rhIL-1 administered to 5-FU treated animals shortened the neutropenic period from 30 to 17 days and increased the number of marrow progenitors responsive to other CSFs. Prolonged administration of IL-1 (14 days) to these animals resulted in a delayed neutrophil recovery as compared to animals receiving short courses of IL-1. rhIL-1 administered to primates receiving marrow grafts after lethal irradiation, did not result in rapid hematopoietic recovery. In humans, studies with CD-34 positive marrow cells showed that IL-1 had a radioprotective effect on a committed and early marrow progenitors. These data show the therapeutic potential of IL-1 in the treatment of chemoradiotherapy induced myelosuppression.     

Cytokine response to strenuous exercise in athletes and non-athletes--an adaptive response

Exercise and physical strenuous activity have been demonstrated to increase the serum TNF-alpha and IL-6. Regular physical training is expected to attenuate such a response. This study was undertaken to understand the impact of regular exercise training on IL-6 and TNF-alpha in athletes and non-athletes. Ten athletes, who have been on regular training for the past 6 months, and 10 age- and sex-matched subjects (non-athlete group) who had no practice of regular exercise, were recruited. Both were subjected to undergo the same frequency level of strenuous exercise. Blood samples were collected; one before strenuous exercise and the other after the exercise. Plasma cytokines, IL-6 and TNF-alpha, were estimated using Sandwich ELISA method. All participants in the study were male with the athletes' age being 18.00+/-1.3years (mean+/-SD) and the non-athletes were aged 20.00+/-0.6years (mean+/-SD). Majority of the athletes and non-athletes demonstrated a rise in IL-6 and a fall in TNF-alpha levels. Further, the athletes showed a lesser magnitude of change in the cytokine levels following a longer duration of exercise than non-athletes. Athletes appear to have an attenuated cytokine response. Regular physical training has been demonstrated to attenuate the immune response to exercise in either direction.     

Carbon dioxide storage capacity of endurance and sprint-trained athletes in exercise

The purpose of this study was to compare CO2 storage capacity of endurance and sprint-trained athletes during steady state exercise. Ten subjects, five sprinters and five distance runners, performed a submaximal treadmill exercise at two different work rates, 45% and 65% of VO2max. CO2 storage capacity was determined by measuring the excess CO2 washout associated with hyperventilation, normalized for body weight and expressed per unit change in mixed venous PCO2 (ml kg-1 Torr-1). Mixed venous PCO2 (PvCO2) was measured by rebreathing equilibration. It was found that CO2 storage capacities of the runners were significantly (P less than 0.05) greater than the sprinters at the two work rates. The sprinters CO2 storage capacities were 2.69 and 2.14 ml kg-1 Torr-1 at low and high work rates, respectively. The corresponding mean values for the runners were 4.56 and 3.92 ml kg-1 Torr-1, respectively. These results may be explained by the metabolic differences between the sprinters and runners. The sprinters' musculature depends more heavily on the glycolytic metabolic pathway, which is associated with an increased lactate production and hence a reduction in the combining power of the blood for CO2 during exercise. At the low work rate, the body's storage capacity for CO2 was significantly (P less than 0.05) greater than the higher work rate for both groups. Obviously, at the higher work level more blood would be presented to the lungs per unit time allowing an increase in CO2 clearance from the body stores.