Hector Mayani

BIOLOGY OF HUMAN UMBILICAL CORD BLOOD-DERIVED HEMATOPOIETIC STEM/PROGENITOR CELLS

Reported in 1989, studies by Broxmeyer, Gluckman, and colleagues demonstrated that umbilical cord blood (UCB) is a rich source of hematopoietic stem/progenitor cells (HSPC) and that UCB could be used in clinical settings for hematopoietic cell transplantation. Since then, a great interest has been generated on the biological characterization of these cells. Over the last nine years, several groups have focused on the study of UCB HSPC, addressing different aspects, such as the frequency of these cells in UCB, the identification of different HSPC subsets based on their immunophenotype, their ability to respond to hematopoietic cytokines, the factors that control their proliferation and expansion potentials, and their capacity to reconstitute hematopoiesis in animal models. Most of these studies have shown that significant functional differences exist between HSPC from UCB and adult bone marrow (i.e., the former possess higher proliferation and expansion potential than the latter). It is also noteworthy that genetic manipulation of UCB HSPC has been achieved by several groups and that genetically modified UCB cells have already been used in the clinic. In spite of the significant advances in the characterization of these cells, we are still in the process of trying to fully understand their biology, both at the cellular and the molecular levels. In the present article, we describe and discuss what is currently known about the biology of UCB HSPC.

In vitro characterization of hematopoietic microenvironment cells from patients with myelodysplastic syndrome.

In vitro studies on the functional integrity of the hematopoietic microenvironment in myelodysplasia have been controversial. Although some of them suggest that such a microenvironment is functionally normal, there is increasing evidence indicating that there are alterations in the function of microenvironment (adherent) cell layers from myelodysplastic syndromes (MDS) marrow. Adherent cell layers developed in vitro, however, consist of a mixture of different cell types-mostly fibroblasts and macrophages-thus, it is not clear which cell type(s) is(are) functionally abnormal in this disorder. In order to address this issue, in the present study, we first assessed some functional properties of MDS-derived adherent cell layers, as a whole, and then we analyzed those same functional properties after separating these cells into two different populations: a fibroblast-enriched cell layer and a macrophage-enriched cell layer. When whole adherent layers from MDS patients were analyzed, no significant differences were observed, as compared to their normal counterparts, in terms of morphology and total cell number. A major difference, however, was observed when analyzing the production of the cytokines interleukin-6 (IL-6) and tumor necrosis factor (TNF-alpha). Indeed, adherent layers from MDS patients produced higher levels of these cytokines (2- and 22-fold, respectively), as compared to normal layers. When fibroblast- and macrophage-enriched cell layers were analyzed, a higher apoptotic index was observed in those derived from MDS marrow (4% of TUNEL-positive cells in normal fibroblast layers versus 27% in MDS-derived fibroblast layers; 7% of TUNEL-positive cells in normal macrophage layers versus 24% in MDS macrophage layers). Macrophages from MDS marrow produced significantly higher levels of TNF-alpha (nine-fold) than their normal counterparts. MDS-derived fibroblasts, on the other hand, produced higher levels of IL-6 (nine-fold), as compared to normal fibroblasts. Surprisingly, whereas normal fibroblasts showed a discrete production of TNF-alpha, we found a very high production of this cytokine in cultures of fibroblasts from MDS patients. In summary, in the present study we have demonstrated that, at least in vitro, both fibroblasts and macrophages from MDS bone marrow (BM) are functionally abnormal. Such abnormalities include an increased apoptotic index, as well as a high production of both IL-6 and TNF-alpha.

Characterization of the Adherent Cells Developed in Dexter‐Type Long‐Term Cultures from Human Umbilical Cord Blood

We have previously shown that when human umbilical cord blood (UCB) cells are cultured in standard Dexter‐type long‐term cultures (D‐LTC), adherent cells develop forming a discrete net on the bottom of the culture flask. The identity of such cells, however, has not been defined. Accordingly, the major goal of the present study was to characterize the adherent cells developed in standard UCB D‐LTC. Cultures were established from 14 UCB samples and from nine bone marrow (BM) samples, as controls. Both UCB and BM cultures were initiated with the same number of mononuclear cells (MNC) (2.5 × 106 MNC/ml). After three weeks in culture, adherent cell numbers in UCB D‐LTC were 24%‐30% of the numbers found in BM cultures. More than 90% of the adherent cells in UCB D‐LTC expressed the acid phosphatase enzyme, whereas no alkaline phosphatase‐positive cells were observed. This was in contrast to BM D‐LTC, in which alkaline and acid phosphatase were expressed by 60%‐75% and 20%‐45% of the adherent cells, respectively. Immunochemical analysis showed that CD61 (osteoclast marker) and Factor VIII (endothelial cell marker) were not expressed by the adherent cells developed in UCB cultures. Interestingly, the majority of such cells expressed CD1a (dendritic cell marker), CD14, CD68 and CD115 (antigens mainly expressed by macrophagic cells). When the cultures were supplemented with the recombinant cytokines epidermal growth factor, basic fibroblast growth factor, platelet‐derived growth factor or granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), only GM‐CSF had a significant positive effect on adherent cell number. In order to test for some functional properties of the adherent cells developed in culture, production of stem cell factor (SCF), interleukin 6 (IL‐6) and tumor necrosis factor‐α (TNF‐α) was assessed. IL‐6 and TNF‐α showed elevated levels in UCB D‐LTC, whereas SCF levels were always below detection. Finally, analysis of fibroblast progenitors (fibroblast colony‐forming units [CFU‐F]) showed that these cells were present in BM samples (6 CFU‐F/105 MNC) and were totally absent in UCB samples. Taken together, the results of the present study indicate that the vast majority of the adherent cells developed in standard UCB D‐LTC belong to the macrophage lineage and that fibroblasts seem to be absent. Interestingly, the high proportion of CD1a+ cells suggests that dendritic cells are also present in these cultures.

Functional analysis of myelodysplastic syndromes-derived mesenchymal stem cells.

Two different reports, including one from our own group, have recently demonstrated the presence of severe chromosomal abnormalities in mesenchymal stem cells (MSC) from patients with myelodysplastic syndromes (MDS). In the present study, we have assessed whether such cytogenetic abnormalities result in functional deficiencies in vitro. We found that both normal and MDS MSC showed similar expression patterns of cell adhesion molecules and extracellular matrix proteins. MDS MSC layers showed the capability to differentiate towards adipocytes, chondrocytes and osteoblasts, and supported the growth of early umbilical cord blood progenitors in a co-culture system. Unstimulated MDS MSC secreted more IL-1beta and after treatment with TNFalpha, they secreted more SCF, as compared to their normal counterparts. The present study demonstrates that, in spite of harboring severe chromosomal alterations, most of the functional properties of MDS-derived MSC remain normal, including their ability to support normal hematopoiesis in vitro.

In Vitro Proliferation, Expansion, and Differentiation of a CD34+ Cell-Enriched Hematopoietic Cell Population from Human Umbilical Cord Blood in Response to Recombinant Cytokines

BACKGROUND The conditions and mechanisms that control the in vitro growth of hematopoietic stem/progenitor cells (contained within the population of CD34+ cells) are still not completely understood. METHODS By using an immunomagnetic system, we have enriched for umbilical cord blood (UCB)-derived CD34+ cells (55% of total cells recovered vs. 0.8% of total cells prior to the enrichment procedure) and analyzed their in vitro growth (proliferation, expansion, and differentiation) in a liquid culture system in the absence or presence of different recombinant cytokine combinations. RESULTS When the selected cells were cultured in the absence of recombinant cytokines, no proliferation or expansion was observed. In the presence of steel factor (SF) and interleukin-6 (IL-6), total cell number was increased nearly fourfold; however, no progenitor cell expansion took place. When cultures were supplemented with SF and IL-6 together with IL-3 and erythropoietin (EPO), a rapid proliferation of the CD34+ -enriched cell population was observed with a selective stimulation of erythropoiesis. However, this stimulation was only transient, suggesting that there was a rapid exhaustion of erythroid progenitor cells within the first 10 days. Significantly higher levels of proliferation and expansion of progenitor cells were observed in the presence of SF, IL-6, GM-CSF, and G-CSF with preferential stimulation of myelopoiesis. Interestingly, such stimulation of myelopoiesis was sustained for the entire culture period (>30 days). The highest levels of proliferation and expansion were observed in the presence of all six cytokines. Under these conditions, erythropoiesis was also sustained only transiently (10 days), whereas myelopoiesis was sustained for >30 days. CONCLUSIONS This study indicates that significant proliferation and expansion of hematopoietic progenitors can be achieved in vitro when culturing a cell population in which CD34+ cells comprise only >50% of the total cells. Our results also suggest that myeloid progenitors (those responding to GM-CSF and G-CSF) possess higher expansion potentials in vitro than their erythroid counterparts. The methods described here for the enrichment and culture of CD34+ cells may be relevant in the development of protocols for the ex vivo proliferation and expansion of hematopoietic progenitors for transplantation.

Kinetics of Hematopoiesis in Dexter-Type Long-Term Cultures Established from Human Umbilical Cord Blood Cells

In the present study, we have established Dexter‐type long‐term cultures (D‐LTC) from human umbilical cord blood (UCB) and followed the kinetics of different hematopoietic progenitor cells (HPCs)—including multipotent (colony forming unit [CFU]‐ Mixture), erythroid (CFU‐erythroid, BFU‐E), and myeloid (CFU‐granulocyte, CFU‐macrophage, CFU‐granulocyte/macrophage) progenitors as well as of morphologically recognizable erythroid, myeloid and lymphoid cells—during a nine‐week culture period. D‐LTC were also established from adult bone marrow (BM) as controls. On day 0, both UCB and BM showed similar total numbers of HPCs (about 310/105 cells), however, UCB showed a higher proportion of primitive HPCs (i.e., CFU‐Mixture, CFU‐granulocyte/macrophage and BFU‐E). A poor adherent cell layer, consisting almost exclusively of macrophages, was developed in UCB D‐LTC and this correlated with a continuous decline in HPC numbers throughout the culture period. In contrast, adherent cell numbers in BM D‐LTC, including fibroblasts and macrophages, were two‐ to fourfold higher than in UCB cultures, and the numbers of HPCs were also significantly higher, reaching plateau levels between weeks 6 and 9. In both types of cultures, erythroid and multipotent progenitors declined relatively fast, reaching undetectable levels after five weeks of culture. Myeloid progenitors, on the other hand, were sustained longer (always at higher levels in BM cultures) and were still detected by week 9. Among myeloid progenitors, a shift towards the predominance of macrophage HPCs was observed, both in UCB and BM D‐LTC, and this correlated with an increase in the proportion of mature monocytes and macrophages. Taken together, our results indicate that myeloid progenitor cell growth is deficient in UCB D‐LTC and suggest that this is due to the impaired development of an adherent cell layer, unable to provide the factors and conditions required for their growth. Interestingly, throughout the culture period the total numbers of multipotent and erythroid progenitors were similar both in UCB and BM cultures regardless of the number and types of adherent cells present; this suggests that the stroma developed in D‐LTC is not sufficient for the proliferation of these progenitor cells.

Concise Review: Ex Vivo Expansion of Cord Blood-Derived Hematopoietic Stem and Progenitor Cells: Basic Principles, Experimental Approaches, and Impact in Regenerative Medicine

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) play key roles in the production of mature blood cells and in the biology and clinical outcomes of hematopoietic transplants. The numbers of these cells, however, are extremely low, particularly in umbilical cord blood (UCB); thus, ex vivo expansion of human UCB‐derived HSCs and HPCs has become a priority in the biomedical field. Expansion of progenitor cells can be achieved by culturing such cells in the presence of different combinations of recombinant stimulatory cytokines; in contrast, expansion of actual HSCs has proved to be more difficult because, in addition to needing recombinant cytokines, HSCs seem to deeply depend on the presence of stromal cells and/or elements that promote the activation of particular self‐renewal signaling pathways. Hence, there is still controversy regarding the optimal culture conditions that should be used to achieve this. To date, UCB transplants using ex vivo‐expanded cells have already been performed for the treatment of different hematological disorders, and although results are still far from being optimal, the advances are encouraging. Recent studies suggest that HSCs may also give rise to nonhematopoietic cells, such as neural, cardiac, mesenchymal, and muscle cells. Such plasticity and the possibility of producing nonhematopoietic cells at the clinical scale could bring new alternatives for the treatment of neural, metabolic, orthopedic, cardiac, and neoplastic disorders. Once standardized, ex vivo expansion of human HSCs/HPCs will surely have a positive impact in regenerative medicine.

Design, characterization and application of a minibioreactor for the culture of human hematopoietic cells under controlled conditions

The in vitro culture of human hematopoietic cells has recently received considerable attention due to its clinical importance. Most studies of the culture and expansion of hematopoietic cells have been performed in static cultures but only very few reports exist on the use of bioreactors where strict control of environmental variables is maintained. In this work, the design, characterization and application of a fully instrumented minibioreactor for the culture of human hematopoietic cells from umbilical cord blood is presented. The system consists of a stirred- tank reactor where cells are maintained in suspension in an homogeneous environment and without the need of a stromal feeding layer. The minibioreactor was coupled to a data acquisition and control system which continuously monitored pH, dissolved oxygen and redox potential. When operated at 75 rpm with a hanging magnetic bar (impeller-to-tank diameter ratio of 0.57), the dead and mixing times were 120 and 80 s, respectively, and the maximum response rate and volumetric oxygen transfer coefficient were 0.8 mM O2 hr-1, and 1.8 hr-1, respectively. Such characteristics allowed a tight control of pH(until day 11) and dissolved oxygen at predetermined set-points, and up to a 7-fold expansion of hematopoietic progenitors was possible in cultures maintained at 20% dissolved oxygen with respect to air saturation. Growth phase and cell concentration could be inferred on- line through determinations of oxygen uptake rate and culture redox potential. Oxygen uptake rate increased during exponential growth phase to a maximum of 40 μM hr-1. Such an increase closely followed the increase in concentration of hematopoietic progenitors. In contrast, culture redox potential decreased during exponential growth phase and then increased during death phase. The designed system permits not only the maintenance of controlled environmental conditions and on-line identification of fundamental culture parameters, but also the application of control strategies for improving expansion of hematopoietic cells.

Hematopoietic progenitor cells from patients with myelodysplastic syndromes: in vitro colony growth and long-term proliferation.

It is known that the levels of hematopoietic progenitor cells (HPC) are greatly reduced in the majority of patients with myelodysplastic syndromes (MDS). To date, however, only limited information exists on the growth kinetics of these cells in long-term marrow cultures (LTMC), particularly in terms of erythroid and multipotent progenitors. In the present study, we have determined the HPC content in the bone marrow of 12 MDS patients and followed the proliferation kinetics of myeloid (including granulocyte, macrophage and granulocyte macrophage), erythroid (including early and late) and multipotent progenitor cells in LTMC throughout a 7-week culture period. Both the non-adherent and adherent fractions of the cultures were analyzed, so we were able to look at progenitor cells in suspension and those that physically associated to the stromal cell layer developed in culture. All 12 patients were grouped based on their FAB subtype and the in vitro growth of the HPC was analyzed accordingly. The results presented here indicate that in the majority of MDS patients, pronounced deficiencies exist both in the content and the long-term proliferation of marrow HPC. Such deficiencies were particularly evident for multipotent progenitors and those committed to the erythroid lineage, in which alterations in the maturation process also seem to be present. Our results suggest that, at least in some patients, HPC--besides showing an impaired proliferative capacity--lose their ability to adhere to the stromal cell layers developed in culture. RA patients showed the less affected in vitro HPC growth, whereas HPC from RAEB and RAEB-t showed a markedly deficient growth in culture. Interestingly, myelopoiesis was significantly increased in cultures of CMML patients. These results give some new insights into the biology of MDS-derived HPC.

In vitro biology of human myeloid leukemia

For about 40 years, the biology of human myeloid leukemia (ML) has been studied in different in vitro systems. Throughout this time, semisolid colony assays, Dexter-type long-term cultures and liquid suspension cultures have contributed to our understanding of the mechanisms involved in the origin and progression of this hematological disorder. By using such systems, it has been possible to identify the cells in which leukemia originates; to recognize a functional hierarchy within the hematopoietic system of leukemia patients; to identify factors, soluble and cell-associated, that regulate leukemic growth; and to study the effects of different antineoplastic drugs. Furthermore, in vitro systems for purging of leukemic cells have been developed. Still, many questions and problems remain unsolved regarding the biology of myeloid leukemia in vitro. This article presents a comprehensive review on the behavior of leukemic stem and progenitor cells, both from acute and chronic myeloid leukemia, in the different culture systems mentioned above.