Hematopoiesis and hematopoietic organs in arthropods

Hemocytes (blood cells) are motile cells that move throughout the extracellular space and that exist in all clades of the animal kingdom. Hemocytes play an important role in shaping the extracellular environment and in the immune response. Developmentally, hemocytes are closely related to the epithelial cells lining the vascular system (endothelia) and the body cavity (mesothelia). In vertebrates and insects, common progenitors, called hemangioblasts, give rise to the endothelia and blood cells. In the adult animal, many differentiated hemocytes seem to retain the ability to proliferate; however, in most cases investigated closely, the bulk of hemocyte proliferation takes place in specialized hematopoietic organs. Hematopoietic organs provide an environment where undifferentiated blood stem cells are able to self-renew, and at the same time generate offspring that differentiate into different blood cell types. Hematopoiesis in vertebrates, taking place in the bone marrow, has been subject to intensive research by immunologists and stem cell biologists. Much less is known about blood cell formation in invertebrate animals. In this review, we will survey structural and functional properties of invertebrate hematopoietic organs, with a main focus on insects and other arthropod taxa. We will then discuss similarities, at the molecular and structural level, that are apparent when comparing the development of blood cells in hematopoietic organs of vertebrates and arthropods. Our comparative review is intended to elucidate aspects of the biology of blood stem cells that are more easily missed when focusing on one or a few model species.     

Kurloff cells in peripheral blood and organs of wild capybaras

Peripheral blood and tissue from twenty-two free-ranging, hunter-killed capybaras (Hydrochaeris hydrochaeris) collected between December 1996 and April 1997 in Casanare, Colombia (5 degrees 58'N and 71 degrees 33'W), were examined by light microscopy for Kurloff cells (KCs). Kurloff cells were observed in the blood of one pregnant adult female, and in organs from all the animals, including spleen (21 of 22 animals), liver (18 of 21), lungs (13 of 21), ovary (8 of 11), uterus (7 of 10), bone marrow (13 of 20), kidney (8 of 22), adrenal gland (6 of 20), and lymph node (4 of 14). The anatomic distribution of the KC in the wild capybaras was similar to that of the guinea pig.     

UC blood hematopoietic stem cells and therapeutic angiogenesis

Studies suggest that mobilized hematopoietic stem cells (HSC) are recruited to ischemic tissue and stimulate angiogenesis. Critical observations in pre-clinical studies have identified an augmentation of endogenous microvascular collateralization that is beyond that directly attributable to anatomic incorporation and differentiation of infused human cells into the vascular endothelium. Evidence links age-associated reductions in the levels of circulating marrow-derived HSC characterized by expression of early HSC markers CD133 and CD34, with the occurrence of cardiovascular events and associated death. Utilizing the patient's own HSC to augment angiogenesis has several disadvantages, including reduced function of these cells and logistical issues related to cell collection from individual patients. Thus an available source of allogeneic HSC such as UC blood for cellular therapy may be optimal.     

Nonadhesive populations in cultures of mesenchymal stromal cells from hematopoietic organs in mouse and rat

The study of adhesive properties of multipotent mesenchymal stromal cells evaluated from fibroblast colony-forming units in the bone marrow of adult mice and rats in populations of cells attached and unattached to plastic substrate after 2 h to 7 days in culture demonstrated both similarities and differences. The increase in the fibroblast colony-forming units in the adhesive population peaked on day 7 of in vitro culture in both cases; however, nearly no fibroblast colony-forming units were observed in the nonadhesive population from the mouse bone marrow in this period. Conversely, the number of colonies from the rat bone marrow nonadhesive population on day 7 of culture considerably increased, and this nonadhesive population in long-term culture became the source for subsequent nonadhesive subpopulations containing fibroblast colony-forming units. After 7 days of in vitro culture, the suspension of cells isolated from the liver of 17-day-old rat fetuses also contained a fraction of unattached fibroblast colony-forming units. In the nonadhesive subpopulations from the bone marrow and fetal liver, fibroblast colony-forming units were observed up to day 48 and 30, respectively. Stromal cell precursors of nonadhesive subpopulations from the rat bone marrow featured a period of colony formation reduced to 7 days (i.e., they were formed 1.5-2 times faster compared to the primary culture). The total number of fibroblast colony-forming units from all nonadhesive subpopulations was roughly 6 and 7.4 times that of the adhesive population of the primary culture from the bone marrow and fetal liver, respectively. Considering that the mammalian bone marrow remains the preferred source of mesenchymal stromal cells, using nonadhesive subpopulations in the presented culture system can considerably increase the yield of stromal precursor cells     

Phosphofructokinase isozymes from human organs and blood cells.

Phosphofructokinase (PFK) isozymes of blood cells and some human tissues were studied by starch gel electrophoresis and immunoprecipitation by anti-muscle and anti-erythrocyte PFK sera. PFK from muscle, heart, brain and placenta were totally precipitated by both antisera. PFK from blood cells (erythrocytes, lymphocytes, granulocytes, platelets) were precipitated more strongly by anti-erythrocyte PFK serum than by anti-muscle PFK serum. Liver, kidney and monoblast PFK were slightly precipitated by both antisera. From the electrophoretic patterns and the immunoprecipitation curves we may conclude that muscle contains the homotetrameric M4 forms; platelet, liver and kidney the homotetrameric E4 form, and blood cells the M-E hybrids. Monoblasts probably contain a E4 type PFK precursor, and heart, placenta and brain, a modified M4 type PFK. Other isozymes, unrelated with muscle and erythrocyte, were revealed in liver and kidney.     

Differentiation of hematopoietic cells in explants of embryonal organs of Rana temporaria L]

The potencies of isolated embryonic hemopoietic organs (pronephros and liver) of Rana temporaria L. to the formation of the foci of hemopoiesis were studied. The pronephros and liver rudiments were explanted at the early developmental stages (late neurula and early tail bud) and cultivated in vivo in the diffusion chambers. The blast hemopoietic elements and differentiated blood cells are found in the explants within 7 to 10 days of cultivation. A suggestion is put forward that the differentiation of hemopoietic cells in the embryonic hemopoietic organs proceeds from the local cells-precursors.     

Characteristics of hematopoietic stem cells of umbilical cord blood

Umbilical cord blood collected from the postpartum placenta and cord is a rich source of hematopoietic stem cells (HSCs) and is an alternative to bone marrow transplantation. In this review we wanted to describe the differences (in phenotype, cytokine production, quantity and quality of cells) between stem cells from umbilical cord blood, bone marrow and peripheral blood. HSCs present in cord blood are more primitive than their counterparts in bone marrow or peripheral blood, and have several advantages including high proliferation. With using proper cytokine combination, HSCs can be effectively developed into different cell lines. This process is used in medicine, especially in hematology.     

Effect of stromal mechanocytes of hematopoietic organs on in vitro formation of antibody producing cells

Cultured rabbit fibroblasts of bone marrow, thymus and spleen origin were added in spleen cell cultures in which the primary antibody response to SRBC was induced. Bone marrow fibroblasts caused strong inhibition of the response; thymus fibroblasts stimulated antibody formation; spleen fibroblasts inhibited the response when added in large amounts otherwise they produced no effect. The stimulation of antibody forming cell response by thymus fibroblasts proved independent of whether fibroblasts were irradiated or not. Bone marrow fibroblasts exhibited suppressive effect on the response predominantly during initial stages of antibody induction. All the 3 types of fibroblasts did not influence cell viability in spleen cells cultures, and were much more effective on addition to cultures of A-deficient spleen cells as compared to full spleen cells.     

Transplantation of hematopoietic stem cells from the peripheral blood

Hematopoietic stem cells can be collected from the peripheral blood. These hematopoietic stem cells (HSC), or better progenitor cells, are mostly expressed as the percentage of cells than react with CD34 antibodies or that form colonies in semi-solid medium (CFU-GM). Under steady-state conditions the number of HSC is much lower in peripheral blood than in bone marrow. Mobilization with chemotherapy and/or growth factors may lead to a concentration of HSC in the peripheral blood that equals or exceeds the concentration in bone marrow. Transplantation of HSC from the peripheral blood results in faster hematologic recovery than HSC from bone marrow. This decreases the risk of infection and the need for blood-product support. For autologous stem-cell transplantation (SCT), the use of peripheral blood cells has completely replaced the use of bone marrow. For allogeneic SCT, on the other hand, the situation is more complex. Since peripheral blood contains more T-lymphocytes than bone marow, the use of HSC from the peripheral blood increases the risk of graft-versus-host disease after allogeneic SCT. For patients with goodrisk leukemia, bone marrow is still preferred, but for patients with high-risk disease, peripheral blood SCT has become the therapy of choice.     

Immunophenotyping of hematopoietic progenitor cells: Comparison between cord blood and adult mobilized blood grafts

AIM:To study the immunophenotype of hematopoietic progenitor cells from cord blood (CB) grafts (n = 39) in comparison with adult apheresis grafts (AG, n = 229) and pre-apheresis peripheral blood (PAPB) samples (n = 908) using flow cytometry analysis.METHODS: First, we performed a qualitative analysis of CD34+ cell sub-populations in both CB and PAPB grafts using the standardized ISHAGE protocol and a wide panel of 20 monoclonal antibodies. Next, we stud-ied some parameters, such as the age of mothers and the weight of newborns, which can influence the qual-ity and the quantity of CD34+ cells from CB. RESULTS: We found that the percentage of apoptotic cells was high in CB in comparison to PAPB (PAPB: 4.6% ± 2.6% vs CB: 53.4% ± 5.2%, P < 0.001). In CB, the weight of newborn and the age of the mother have the influence on CD34+ cells. The follow-up of Ag CD133in the ISHAGE double platform protocol in association with CD45, CD34 and the 7’AAD shows an equal rate between the two cell populations CD133+CD45+CD34+ high and CD34+CD45+ high with a higher percentage. So, is the inclusion of Ac CD133 necessary in the pres-ent panel included in the ISHAGE methodflLast part, we showed a signif icant presence of interferon γ in CB in comparison to PAPB, the annexin showing the high number of apoptotic cells in CB. CONCLUSION: This study demonstrates that many different obstetric factors must be taken into account when processing and cryo-banking umbilical CB units for transplantation.     

Stromal fibroblasts of the hematopoietic organs and antibody formation in cultures

The thymus, bone marrow- and spleen-derived stromal mechanocytes from the monolayer cultures (3rd--6th passages) when added to the suspension cultures of rabbit spleen cells according to Mishell and Dutton produced a considerable effect on the plaque-forming cells (PFC) accumulation by the 4th day of cultivation. Their action distinctly depended on the dose. Bone marrow-derived stromal mechanocytes in doses of 2.1 X 10(3)--6.25 X 10(5) caused inhibition of PFC formation in cultures. Thymus-derived stromal mechanocytes in doses of 2.75 X 10(3)--8 X 10(5) cause an increase in number of PFC; spleen-derived stromal mechanocytes in doses of 2.1 X 10(3)--1.3 X 10(4) failed to bring about any significant changes, but when the dose was increased to 8 X 10(4)--6.25 X 10(5) the inhibition of PFC formation took place. Most of the live cells and PFC were found in the free cells fraction.     

Expansion of human cord blood hematopoietic stem cells for transplantation

A recent Science paper reported a purine derivative that expands human cord blood hematopoietic stem cells in culture (Boitano et al., 2010) by antagonizing the aryl hydrocarbon receptor. Major problems need to be overcome before ex vivo HSC expansion can be used clinically.     

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.