SummaryBlood-derived adult stem cells were previously considered impractical for therapeutic use because of their small numbers. This report describes the isolation of a novel human cell population derived from the peripheral blood, termed synergetic cell population (SCP) Neural cell precursors (NCPs) expressed the neuronal markers Nestin, bIII-Tubulin, and Neu-N, the glial markers GFAP and O4, and responded to neurotransmitter stimulation. Myocardial cell precursors (MCPs) expressed Desmin, cardiac Troponin and Connexin 43. In conclusion, the simple and rapid method of SCP generation and the resulting considerable quantities of lineage-specific precursor cells makes it a potential source of autologous treatment for a variety of diseases.
To generate an experimental model for exploring the function, expression pattern, and developmental regulation of human Ig-like activating and inhibitory receptors, we have generated transgenic mice using two human genomic clones: 52N12 (a 150-Kb clone encompassing the leukocyte Ig-like receptor (LILR)B1 (ILT2), LILRB4 (ILT3), and LILRA1 (LIR6) genes) and 1060P11 (a 160-Kb clone that contains ten killer cell Ig-like receptor (KIR) genes). Both the KIR and LILR families are encoded within the leukocyte receptor complex, and are involved in immune modulation. We have also produced a novel mAb to LILRA1 to facilitate expression studies. The LILR transgenes were expressed in a similar, but not identical, pattern to that observed in humans: LILRB1 was expressed in B cells, most NK cells, and a small number of T cells; LILRB4 was expressed in a B cell subset; and LILRA1 was found on a ring of cells surrounding B cell areas on spleen sections, consistent with other data showing monocyte/macrophage expression. KIR transgenic mice showed KIR2DL2 expression on a subset of NK cells and T cells, similar to the pattern seen in humans, and expression of KIR2DL4, KIR3DS1, and KIR2DL5 by splenic NK cells. These observations indicate that linked regulatory elements within the genomic clones are sufficient to allow appropriate expression of KIRs in mice, and illustrate that the presence of the natural ligands for these receptors, in the form of human MHC class I proteins, is not necessary for the expression of the KIRs observed in these mice.
In the last few years, significant progress has been made in the isolation and characterization of bone marrow stem cell populations and their potential to differentiate into a variety of cellular lineages. We hypothesized that peripheral blood can also be used as a source for precursor cells that can become committed progenitors for a variety of tissues. We report here the generation and characterization in vitro of neural progenitor cells from a newly discovered blood-derived multipotent cell population, named synergetic cell population (SCP). Human blood samples were obtained from the Israeli blood bank and SCP cells were purified based on cellular density. Neural progenitors were generated by culturing SCP cells in medium supplemented with autologous serum, followed by activation in a defined serum-free medium containing the specific differentiation-inducing factors F12, B27, bFGF, BDNF, and NGF. An average of 13.5x106 neural progenitor cells was generated from 450 ml blood. These cells developed irregular perikarya, from which filamentous extensions spread contacting neighboring cells and forming net-like structures. Immunostaining revealed that some of the cells express the early neuronal progenitor markers nestin and b-tubulin and Neu-N, a nuclear protein present in mature neurons. Other cells expressed glial-specific antigens, such as O4 (a marker of oligodendrocytes) and GFAP (a marker of astrocytes). Flow cytometry analysis showed that 44.4% and 34% of the cells were positive for nestin and b-tubulin, respectively. In addition to exhibiting phenotypic evidence of markers specific for the neural lineage, these progenitor cells also responded to the neurotransmitters glutamate and GABA, as detected by calcium influx through voltage-gated calcium channels, demonstrating functional differentiation. In this study we show that generation of neural progenitors from peripheral blood is feasible and efficient. Blood-derived angiogenic progenitors produced in our system are already safely and efficiently administrated to severe angina pectoris patients in a clinical trial we are conducting in Thailand (reported in a separate abstract by our group). The newly discovered source of these progenitors, the blood-derived multipotent population which we termed SCP, contains both hematopoietic stem cells as well as supportive cells that enable differentiation into various lineages. The therapeutic potential of these neural progenitors will be further characterized and evaluated in vivo using animal models.
Significant progress has been made in recent years in developing therapeutic strategies for the treatment of a variety of cardiovascular disorders, mainly using bone marrow-derived progenitor cells. We hypothesized that blood leukocytes can also serve as a source for a wide range of clinical protocols. We report here the generation in vitro of both angiogenic cell precursors (ACP) and cardiomyocyte (CMC) progenitors from a newly discovered blood-derived multipotent cell population, termed synergetic cell population (SCP), and their function in vitro and in vivo. Progenitor cells were purified from healthy donor blood samples using density-based gradients. SCP-derived ACPs grown in the presence of autologous serum and VEGF exhibited an elongated, spindle-shaped morphology and expressed the stem cell markers CD34 (an average of 23.1% of cells), CD133 (10.2%), and CD117 (10.8%), and the endothelial markers KDR (8.9%), Tie-2 (24.8%), CD144 (41.2%), and CD31 (83.1%). Up to 30% of the cells exhibited Dil-Ac-LDL uptake, typical of endothelial cells. In vitro, ACPs showed organization into capillary tube structures when plated on extracellular matrix gels. An average of 50x106 ACPs were generated from 450 ml blood. CMC progenitors, which resulted from culturing SCP cells in medium containing autologous serum and bFGF followed by activation in a medium containing 5-azacytidine, appeared elongated with dark cytoplasm and expressed the cardiomyocyte markers desmin and troponin (on 19.7% and 52.3% of cells, respectively). The therapeutic potential of blood derived ACPs is currently being evaluated in patients with severe angina pectoris. Seventeen patients on maximal drug therapy have so far been prospectively enrolled, based upon identifying ischemic but viable myocardium in distribution of the coronary arteries that were totally occluded. ACPs (25x106, SE=4.9) were injected via a catheter into the coronary artery. Preliminary results demonstrate safety and improved clinical symptoms at 3 months vs. baseline. Mean Canadian Cardiovascular Scale for angina severity decreased from 1.8±0.8 to 1.06±0.3 (P=0.062) and exercise capacity measured by metabolic equivalents increased from 6.3±2.3 to 7.4 ±2.8 (P= 0.0083). One patient died two weeks after the treatment due to acute myocardial infarction. However, coronary angiography demonstrated acute occlusion of an artery not treated with cells. These results suggest the treatment is safe with preliminary short term beneficial effect. Continued follow-up is currently being conducted to determine the long-term effects of this therapy in a larger number of patients. In order to examine the functional mechanisms underlying the therapeutic effects of ACPs and CMC progenitors, an in-vivo experiment is also being carried out in a nude rat acute myocardial infarction model. We demonstrate here that a newly-discovered multipotent cell population which we term SCP can be isolated from peripheral blood and differentiated into therapeutically effective tissue-committed progenitor cells. The SCP contains hematopoietic stem cells and supportive cells enabling differentiation into various lineages, such as ACPs, cardiomyocyte and neural progenitors (the latter reported in a separate abstract by our group) which have thus been generated.
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