Further biological characterization of small molecules UM171 and SR1: In vitro effects on three hematopoietic cell populations from human cord blood.

Small molecules UM171 and SR1 have already been taken into clinically-oriented protocols for the ex vivo expansion of hematopoietic stem (HSCs) and progenitor (HPCs) cells. In order to gain further insight into their biology, in the present study we have assessed their effects, both individually and in combination, on the in vitro long-term proliferation and expansion of HSCs and HPCs contained within three different cord blood-derived cell populations: MNCs (CD34 cells = 0.8 %), LIN cells (CD34 cells = 41 %), and CD34 cells (CD34 cells >98 %).

Isolation of human and murine hematopoietic stem cells for DNA damage and DNA repair assays.

Hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow and supply blood cells. Efficient methods for isolation of HSPCs are required. Here, we present protocols for the isolation of human and murine HSPCs using manual and FACS-assisted techniques.

Inhibition of TGFβ1 and TGFβ3 promotes hematopoiesis in Fanconi anemia.

Fanconi anemia (FA) is a chromosome instability syndrome with congenital abnormalities, cancer predisposition and bone marrow failure (BMF). Although hematopoietic stem and progenitor cell (HSPC) transplantation is the recommended therapy, new therapies are needed for FA patients without suitable donors. BMF in FA is caused, at least in part, by a hyperactive growth-suppressive transforming growth factor β (TGFβ) pathway, regulated by the TGFβ1, TGFβ2, and TGFβ3 ligands.

Human cord blood hematopoietic cells acquire neural features when cultured in the presence of neurogenic cytokines.

In vitro growth of hematopoietic cells depends on the presence of hematopoietic cytokines. To date, it is unclear if these cells would be able to respond to non-hematopoietic cytokines. In the present study, we have explored this by culturing human hematopoietic cells in presence of neurogenic cytokines.

The in vitro growth of a cord blood-derived cell population enriched for CD34 cells is influenced by its cell cycle status and treatment with hydroxyurea.

Objective: Cell cycle plays a fundamental role in the physiology of hematopoietic stem and progenitor cells. In the present study we used a negative selection system to obtain an immature cell population-enriched for cord blood-derived CD34 cells-and we determined its proliferation, expansion and differentiation patterns as a function of the cell cycle status. The effects of hydroxyurea (HU) were also assessed.

Functional Integrity and Gene Expression Profiles of Human Cord Blood-Derived Hematopoietic Stem and Progenitor Cells Generated In Vitro.

To date, different experimental strategies have been developed for the ex vivo expansion of human hematopoietic stem (HSCs) and progenitor (HPCs) cells. This has resulted in significant advances on the use of such expanded cells in transplantation settings. To this day, however, it is still unclear to what extent those stem and progenitor cells generated in vitro retain the functional and genomic integrity of their freshly isolated counterparts.

Tubulin is retained throughout the human hematopoietic/erythroid cell differentiation process and plays a structural role in sedimentable fraction of mature erythrocytes.

We investigated the properties of tubulin present in the sedimentable fraction ("Sed-tub") of human erythrocytes, and tracked the location and organization of tubulin in various types of cells during the process of hematopoietic/erythroid differentiation. Sed-tub was sensitive to taxol/nocodazole (drugs that modify microtubule assembly/disassembly), but was organized as part of a protein network rather than in typical microtubule form. This network had a non-uniform "connected-ring" structure, with tubulin localized in the connection areas and associated with other proteins.

Human Mesenchymal Stem/Stromal Cells from Umbilical Cord Blood and Placenta Exhibit Similar Capacities to Promote Expansion of Hematopoietic Progenitor Cells In Vitro.

Mesenchymal stem/stromal cells (MSCs) from bone marrow (BM) have been used in coculture systems as a feeder layer for promoting the expansion of hematopoietic progenitor cells (HPCs) for hematopoietic cell transplantation. Because BM has some drawbacks, umbilical cord blood (UCB) and placenta (PL) have been proposed as possible alternative sources of MSCs. However, MSCs from UCB and PL sources have not been compared to determine which of these cell populations has the best capacity of promoting hematopoietic expansion.

Bone Marrow Mesenchymal Stromal Cells from Clinical Scale Culture: In Vitro Evaluation of Their Differentiation, Hematopoietic Support, and Immunosuppressive Capacities.

The differentiation capacity, hematopoietic support, and immunomodulatory properties of human bone marrow mesenchymal stromal cells (BM-MSCs) make them attractive therapeutic agents for a wide range of diseases. Clinical scale cultures (CSCs) have been used to expand BM-MSCs for their use in cell therapy protocols; however, little is known about the functionality of the expanded cells. The main goal of the present study was to evaluate the functional characteristics of BM-MSCs expanded from CSCs to determine the quality of the cells for cellular therapy protocols.

Lymphoid progenitor cells from childhood acute lymphoblastic leukemia are functionally deficient and express high levels of the transcriptional repressor Gfi-1.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. Substantial progress on understanding the cell hierarchy within ALL bone marrow (BM) has been recorded in the last few years, suggesting that both primitive cell fractions and committed lymphoid blasts with immature stem cell-like properties contain leukemia-initiating cells. Nevertheless, the biology of the early progenitors that initiate the lymphoid program remains elusive.

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.

Comparative in vitro analysis of different hematopoietic cell populations from human cord blood: in search of the best option for clinically oriented ex vivo cell expansion.

Background: Ex vivo expansion of hematopoietic stem and progenitor cells has become a priority in the experimental hematology arena. In this study we have obtained different hematopoietic cell populations from umbilical cord blood and simultaneously assessed their proliferation and expansion kinetics. Our main goal was to determine which one of these cell populations would be more suitable for clinical-grade ex vivo expansion.

In vitro effects of stromal cells expressing different levels of Jagged-1 and Delta-1 on the growth of primitive and intermediate CD34(+) cell subsets from human cord blood.

In trying to contribute to our knowledge on the role of Notch and its ligands within the human hematopoietic system, we have assessed the effects of the OP9 stroma cell line - naturally expressing Jagged-1 - transduced with either the Delta-1 gene (OP9-DL1 cells) or with vector alone (OP9-V), on the in vitro growth of two different hematopoietic cell populations. Primitive (CD34(+) CD38(-) Lin(-)) and intermediate (CD34(+) CD38(+) Lin(-)) CD34(+) cell subsets from human cord blood were cultured in the presence of 7 stimulatory cytokines under four different conditions: cytokines alone (control); cytokines and mesenchymal stromal cells; cytokines and OP9-V cells; cytokines and OP9-DL1 cells. Proliferation and expansion were determined after 7days of culture.

In vitro growth of hematopoietic progenitors and stromal bone marrow cells from patients with multiple myeloma.

In the present study we have determined the content of hematopoietic and stromal progenitors in multiple myeloma (MM) bone marrow, and assessed their in vitro growth. Marrow cells were obtained from 17 MM patients at the time of diagnosis, and from 6 hematologically normal subjects. When mononuclear cells (MNC) from MM marrow were cultured, reduced numbers of hematopoietic progenitors were detected and their growth in long-term cultures was deficient, as compared to cultures of normal cells.

Individual and combined effects of mesenchymal stromal cells and recombinant stimulatory cytokines on the in vitro growth of primitive hematopoietic cells from human umbilical cord blood.

Background Aims: We have previously characterized the in vitro growth of two cord blood-derived hematopoietic cell populations in liquid cultures supplemented with recombinant cytokines. In the present study, we assessed the effects of bone marrow-derived mesenchymal stromal cells (MSC) on the growth of such cells.

Methods: CD34(+) CD38(+) Lin(-) and CD34(+) CD38(-) Lin(-) cells were obtained by negative selection, and cultured in the presence of marrow-derived MSC and/or early- and late-acting cytokines.

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.

Deficient proliferation and expansion in vitro of two bone marrow cell populations from patients with acute myeloid leukemia in response to hematopoietic cytokines.

One has previously characterized two different hematopoietic cell populations (obtained by negative-selection) from normal bone marrow. Population I was enriched for CD34+ Lin- cells, whereas Population II was enriched for CD34+ CD38- Lin- cells. Both populations showed elevated proliferation and expansion potentials in serum-free liquid cultures, supplemented with a combination of eight different cytokines, with the latter displaying more immature features than the former.

In vitro proliferation and expansion of hematopoietic progenitors from patients with diffuse large B-cell lymphoma before and after chemotherapy.

We have previously demonstrated that when cultured in Dexter-type Long-Term Marrow Cultures (LTMC), hematopoietic progenitor cells (HPC) from patients with Diffuse Large B-Cell Lymphoma (DLBCL) show a defective proliferation, as compared to HPC from normal marrow. In that study it was also demonstrated that functional alterations were present in the hematopoietic microenvironment developed in culture; thus, it was not clear whether such a defective proliferation in vitro was due to an intrinsic defect in the HPC compartment of DLBCL patients, or to an altered microenvironment, or both. In order to address this question, in the present study we have assessed the proliferation and expansion potentials of HPC present in bone marrow from patients with DLBCL, in cytokine-supplemented liquid cultures initiated with a cell population enriched for CD34+ Lin- cells, in the absence of stromal cells and in the presence of reduced numbers of accessory cells.

In vitro proliferation and expansion of hematopoietic progenitors present in mobilized peripheral blood from normal subjects and cancer patients.

In the present study, we have assessed, in a comparative manner, the in vitro proliferation and expansion potentials of hematopoietic progenitor cells (HPC) present in mobilized peripheral blood from normal subjects (MPB-n; n = 18) and cancer patients (MPB-c; n = 18). The latter included patients with breast cancer (BrCa; n = 8), Hodgkin disease (HD; n = 4), non-Hodgkin lymphoma (NHL; n = 3), and acute myeloid leukemia (AML; n = 3). Progenitor cells from normal bone marrow (BM) and umbilical cord blood (UCB) were included as controls.

Biology of human hematopoietic stem and progenitor cells present in circulation.

Circulating hematopoietic stem and progenitor cells play important roles in the physiology and homeostasis of the hematopoietic system. The frequency of these cells varies throughout development, being more abundant during gestation. In the adult, the numbers of such cells are extremely low; however, they can be increased by intravenous administration of chemotherapy and/or recombinant cytokines to individuals.

Comparative analysis of the in vitro proliferation and expansion of hematopoietic progenitors from patients with aplastic anemia and myelodysplasia.

Aplastic anemia (AA) and myelodysplasia (MDS) show great similarities in their biology. To date, however, it is still unclear to what extent hematopoietic progenitor cells (HPCs) from AA and MDS share biological properties and what the functional differences are between them. In trying to address this issue, in the present study we have analyzed, in a comparative manner, the proliferation and expansion capacities of bone marrow (BM) progenitor cells from AA and MDS in response to recombinant cytokines.

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.