Altered neuronal network and rescue in a human MECP2 duplication model.

Increased dosage of methyl-CpG-binding protein-2 (MeCP2) results in a dramatic neurodevelopmental phenotype with onset at birth. We generated induced pluripotent stem cells (iPSCs) from patients with the MECP2 duplication syndrome (MECP2dup), carrying different duplication sizes, to study the impact of increased MeCP2 dosage in human neurons. We show that cortical neurons derived from these different MECP2dup iPSC lines have increased synaptogenesis and dendritic complexity. In addition, using multi-electrodes arrays, we show that neuronal network synchronization was altered in MECP2dup-derived neurons. Given MeCP2 functions at the epigenetic level, we tested whether these alterations were reversible using a library of compounds with defined activity on epigenetic pathways. One histone deacetylase inhibitor, NCH-51, was validated as a potential clinical candidate. Interestingly, this compound has never been considered before as a therapeutic alternative for neurological disorders. Our model recapitulates early stages of the human MECP2 duplication syndrome and represents a promising cellular tool to facilitate therapeutic drug screening for severe neurodevelopmental disorders.

Scalable expansion of multipotent adult progenitor cells as three-dimensional cell aggregates.

Many applications of stem cell technologies require a large quantity of cells for which scalable processes of cell expansion and differentiation are essential. Multipotent adult progenitor cells (MAPCs) are adult stem cells isolated from the bone marrow with extensive self-renewal and broad differentiation capabilities. MAPCs are typically cultured surface adherent (2D) and at low cell density, making the large surface required for cell expansion a hindrance for many applications. This study demonstrates that MAPCs can be cultivated as aggregates in an undifferentiated state for at least 16 days, as levels of a number of transcripts, including Oct4, remained similar, Oct4 protein was unchanged, and differentiation to neural progenitor, endothelial cell and hepatocyte like cells was retained. Cultivation of these aggregates in stirred bioreactor lead to a 70-fold expansion in 6 days with final cell densities of close to 106/mL. Importantly, the MAPC aggregates recovered from stirred bioreactors could be differentiated to hepatocyte-like cells that expressed albumin, alpha-1-antitrypsin (AAT), and tyrosine amino transferase (TAT) transcripts and also secreted albumin and urea. This method of scalable expansion combined with differentiation of MAPCs can potentially be used for generating large numbers of MAPC and MAPC-derived differentiated cells.

Expansion and hepatic differentiation of rat multipotent adult progenitor cells in microcarrier suspension culture.

Many potential applications of stem cells require large quantities of cells, especially those involving large organs such as the liver. For such applications, a scalable reactor system is desirable to ensure a reliable supply of sufficient quantities of differentiation competent or differentiated cells. We employed a microcarrier culture system for the expansion of undifferentiated rat multipotent adult progenitor cells (rMAPC) as well as for directed differentiation of these cells to hepatocyte-like cells. During the 4-day expansion culture, cell concentration increased by 85-fold while expression level of pluripotency markers were maintained, as well as the MAPC differentiation potential. Directed differentiation into hepatocyte-like cells on the microcarriers themselves gave comparable results as observed with cells cultured in static cultures. The cells expressed several mature hepatocyte-lineage genes and asialoglycoprotein receptor-1 (ASGPR-1) surface protein, and secreted albumin and urea. Microcarrier culture thus offers the potential of large-scale expansion and differentiation of stem cells in a more controlled bioreactor environment.

Stem and progenitor cells for liver repopulation: can we standardise the process from bench to bedside?

There has been recent progress in the isolation and characterisation of stem/progenitor cells that may differentiate towards the hepatic lineage. This has raised expectations that therapy of genetic or acquired liver disease might be possible by transplanting stem/progenitor cells or their liver-committed progeny. However, it is currently impossible to determine from the many documented studies which of the stem/progenitor cell populations are the best for therapy of a given disease. This is largely because of the great variability in methods used to characterise cells and their differentiation ability, variability in transplantation models and inconsistent methods to determine the effect of cell grafting in vivo. This manuscript represents a first proposal, created by a group of investigators ranging from basic biologists to clinical hepatologists. It aims to define standardised methods to assess stem/progenitor cells or their hepatic lineage-committed progeny that could be used for cell therapy in liver disease. Furthermore standardisation is suggested both for preclinical animal models to evaluate the ability of such cells to repopulate the liver functionally, and for the ongoing clinical trials using mature hepatocytes. Only when these measures have been put in place will the promise of stem/progenitor-derived hepatocyte-based therapies become reality.

BCR/ABL-mediated downregulation of genes implicated in cell adhesion and motility leads to impaired migration toward CCR7 ligands CCL19 and CCL21 in primary BCR/ABL-positive cells.

The mechanism underlying p210(BCR/ABL) oncoprotein-mediated transformation in chronic myelogenous leukemia (CML) is not fully understood. We hypothesized that p210(BCR/ABL) suppresses expression of genes which may explain at least some of the pathogenetic features of CML. A subtractive cDNA library was created between BCR/ABL-enhanced-green-fluorescent-protein (GFP)-transduced umbilical cord blood (UCB) CD34+ cells and GFP-transduced UCB CD34+ cells to identify genes whose expression is downregulated by p210(BCR/ABL). At least 100 genes were identified. We have confirmed for eight of these genes that expression was suppressed by quantitative real-time-RT-PCR (Q-RT-PCR) of additional p210(BCR/ABL)-transduced CD34+ UCB cells as well as primary early chronic phase (CP) bone marrow (BM) CML CD34+ cells. Imatinib mesylate reversed downregulation of some genes, to approximately normal levels. Several of the genes are implicated in cell adhesion and motility, including L-selectin, intercellular adhesion molecule-1 (ICAM-1), and the chemokine receptor, CCR7, consistent with the known defect in adhesion and migration of CML cells. Compared with GFP UCB or normal (NL) BM CD34+ cells, p210 UCB and CML CD34+ cells migrated poorly towards the CCR7 ligands, CCL19 and CCL21, suggesting a possible role for CCR7 in the abnormal migratory behavior of CML CD34+ cells.

p210BCR/ABL-induced alteration of pre-mRNA splicing in primary human CD34+ hematopoietic progenitor cells.

Chronic myelogenous leukemia (CML) is a malignancy of the human hematopoietic stem cell (HSC) caused by the p210BCR/ABL oncoprotein. Although alternative splicing of pre-mRNA is a critical determinant of a cell's protein repertoire, it has not been associated with CML pathogenesis. We identified a BCR/ABL-dependent increase in expression of multiple genes involved in pre-mRNA splicing (eg SRPK1, RNA Helicase II/Gu, and hnRNPA2/B1) by subtractive hybridization of cDNA from p210BCR/ABL-eGFP vs eGFP-transduced umbilical cord blood CD34+ cells. beta1-integrin signaling is important to HSC maintenance and proliferation/differentiation, and is abnormal in CML. As an example of how changes in pre-mRNA processing might contribute to CML pathogenesis, we observed alternative splicing of a gene for a beta1-integrin-responsive nonreceptor tyrosine kinase (PYK2), resulting in increased expression of full-length Pyk2 in BCR/ABL-containing cells. Treatment of p210BCR/ABL-positive cells with the Abl-specific tyrosine kinase inhibitor STI571 reverted PYK2 splicing to a configuration more consistent with normal cells, and correlated with decreased expression of BCR/ABL-induced proteins involved in pre-mRNA processing. Whether altered PYK2 splicing contributes to CML pathogenesis remains undetermined; however, we propose that generic changes in pre-mRNA splicing as a result of p210BCR/ABL kinase activity may contribute to CML pathogenesis.

[Adult stem cells]. / Células madre adultas.

One of the fields of medicine that has created the greatest expectations in recent years is cellular therapy with stem cells. The isolation of human embryo cells, the apparent and unexpected potential of adult stem cells, and the development of gene therapy lead us to imagine a hopeful future for a significant number of diseases that are at present incurable. In the following pages we offer a sketch of the panorama of research with stem cells, describing the main achievements in this field as well as some of the questions awaiting answers. In spite of the great expectations, it is essential that we maintain a critical and realistic spirit when it comes to analysing the scientific advances in this area.

Chronic myelogenous leukemia: mechanisms underlying disease progression.

Chronic myelogenous leukemia (CML), characterized by the BCR-ABL gene rearrangement, has been extensively studied. Significant progress has been made in the area of BCR-ABL-mediated intracellular signaling, which has led to a better understanding of BCR-ABL-mediated clinical features in chronic phase CML. Disease progression and blast crisis CML is associated with characteristic non-random cytogenetic and molecular events. These can be viewed as increased oncogenic activity or loss of tumor suppressor activity. However, what causes transformation and disease progression to blast crisis is only poorly understood. This is in part due to the lack of a good in vivo model of chronic phase CML even though animal models developed over the last few years have started to provide insights into blast crisis development. Thus, additional in vitro and in vivo studies will be needed to provide a complete understanding of the contribution of BCR-ABL and other genes to disease progression and to improve therapeutic approaches for blast crisis CML.

Leukemia in donor cells after allogeneic hematopoietic stem cell transplant.

The development of leukemia in donor cells after allogeneic hematopoietic stem cell transplant is an extremely rare event. We report here the case of a patient who developed myelodysplastic syndrome/acute myeloid leukemia, in cells of donor origin 3.5 years after related donor HSCT for refractory chronic lymphocytic leukemia and therapy-induced myelodysplastic syndrome. The origin of the leukemia was determined by analysis of minisatillite polymorphism tested on CD34(+) cells.

Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells.

It is here reported that mesenchymal stem cells known to give rise to limb-bud mesoderm can, at the single-cell level, also differentiate into cells of visceral mesoderm and can be expanded extensively by means of clinically applicable methods. These cells were named mesodermal progenitor cells (MPCs). MPCs were selected by depleting bone marrow mononuclear cells from more than 30 healthy human donors of CD45(+)/glycophorin-A (GlyA)(+) cells. Cells were cultured on fibronectin with epidermal growth factor and platelet-derived growth factor BB and 2% or less fetal calf serum. It was found that 1/5 x 10(3) CD45(-)GlyA(-) cells, or 1/10(6) bone marrow mononuclear cells, gave rise to clusters of small adherent cells. Cell-doubling time was 48 to 72 hours, and cells have been expanded in culture for more than 60 cell doublings. MPCs are CD34(-), CD44(low), CD45(-), CD117 (cKit)(-), class I-HLA(-), and HLA-DR(-). MPCs differentiated into cells of limb-bud mesoderm (osteoblasts, chondrocytes, adipocytes, stroma cells, and skeletal myoblasts) as well as visceral mesoderm (endothelial cells). Retroviral marking was used to definitively prove that single MPCs can differentiate into cells of limb bud and visceral mesoderm. Thus, MPCs that proliferate without obvious senescence under clinically applicable conditions and differentiate at the single-cell level not only into mesenchymal cells but also cells of visceral mesoderm may be an ideal source of stem cells for treatment of genetic or degenerative disorders affecting cells of mesodermal origin.

Characterization of multipotent adult progenitor cells, a subpopulation of mesenchymal stem cells.

Mesenchymal stem cells were isolated and a subpopulation of cells--multipotent adult progenitor cells--were identified that have the potential for multilineage differentiation. Their ability to engraft and differentiate in vivo is under investigation.

Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system.

This report describes stroma-based and stroma-free cultures that maintain long-term engrafting hematopoietic cells for at least 14 days ex vivo. Umbilical cord blood (UCB) CD34(+) cells were cultured in transwells above AFT024 feeders with fetal-liver-tyrosine-kinase (FL) + stem cell factor (SCF) + interleukin 7 (IL-7), or FL + thrombopoietin (Tpo). CD34(+) progeny were transplanted into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice or preimmune fetal sheep. SCID repopulating cells (SRC) with multilineage differentiation potential were maintained in FL-SCF-IL-7 or FL-Tpo containing cultures for up to 28 days. Marrow from mice highly engrafted with uncultured or expanded cells induced multilineage human hematopoiesis in 50% of secondary but not tertiary recipients. Day 7 expanded cells engrafted primary, secondary, and tertiary fetal sheep. Day 14 expanded cells, although engrafting primary and to a lesser degree secondary fetal sheep, failed to engraft tertiary recipients. SRC that can be transferred to secondary recipients were maintained for at least 14 days in medium containing glycosaminoglycans and cytokines found in stromal supernatants. This is the first demonstration that ex vivo culture in stroma-noncontact and stroma-free cultures maintains "long-term" engrafting cells, defined by their capacity to engraft secondary or tertiary hosts. (Blood. 2001;97:3441-3449)

Novel therapies for chronic myelogenous leukemia.

The BCR-ABL oncogene is essential to the pathogenesis of chronic myelogenous leukemia, and immune mechanisms play an important role in control of this disease. Understanding of the molecular pathogenesis of chronic myelogenous leukemia has led to the development of several novel therapies, which can be broadly divided into therapies based on 1) inhibition of the BCR-ABL oncogene expression, 2) inhibition of other genes important to the pathogenesis of chronic myelogenous leukemia, 3) inhibition of BCR-ABL protein function, and 4) immunomodulation. We have systematically reviewed each of these novel therapeutic approaches in this article.

A model of human p210(bcr/ABL)-mediated chronic myelogenous leukemia by transduction of primary normal human CD34(+) cells with a BCR/ABL-containing retroviral vector.

Most insights into the molecular mechanisms underlying transformation by the p210(BCR/ABL) oncoprotein are derived from studies in which BCR/ABL cDNA was introduced into hematopoietic or fibroblast cell lines. However, such cell line models may not represent all the features of chronic myelogenous leukemia (CML) caused by additional genetic abnormalities and differences in the biology of cell lines compared with primary hematopoietic progenitor and stem cells. A primary human hematopoietic progenitor cell model for CML was developed by the transduction of b3a2 BCR/ABL cDNA in normal CD34(+) cells. Adhesion of BCR/ABL-transduced CD34(+) cells to fibronectin was decreased, but migration over fibronectin was enhanced compared with that of mock-transduced CD34(+) cells. Adhesion to fibronectin did not decrease the proliferation of BCR/ABL-transduced CD34(+) cells but decreased the proliferation of mock-transduced CD34(+) cells. This was associated with elevated levels of p27(Kip) in p210(BCR/ABL)-expressing CD34(+) cells. In addition, the presence of p210(BCR/ABL) delayed apoptosis after the withdrawal of cytokines and serum. Finally, significantly more and larger myeloid colony-forming units grew from BCR/ABL than from mock-transduced CD34(+) cells. Thus, the transduction of CD34(+) cells with the b3a2-BCR/ABL cDNA recreates most, if not all, phenotypic abnormalities seen in primary CML CD34(+) cells. This model should prove useful for the study of molecular mechanisms associated with the presence of p210(BCR/ABL) in CML.

Equivalent outcomes in patients with chronic myelogenous leukemia after early transplantation of phenotypically matched bone marrow from related or unrelated donors.

PURPOSE: To determine if the favorable outcomes after transplantation of matched sibling donor bone marrow in patients with chronic myelogenous leukemia can be achieved using bone marrow from an HLA-A,B/DRB1-matched unrelated donor. SUBJECTS AND METHODS: Between April 1983 and December 1997, 141 patients with chronic myelogenous leukemia in its first chronic phase received a bone marrow transplant from a matched sibling donor (n = 96) or an HLA-A,B/DRB1-matched unrelated donor (n = 45). The median age of matched sibling donor recipients was 38 years (range, 8 to 56 years) and of unrelated donor recipients was 35 years (range, 3 to 53 years; P = 0.03). The median follow-up was 6 years (range, 1 to 15 years) in matched sibling donor recipients and 5 years (range, 2 to 10 years) in unrelated donor recipients. RESULTS: There was no significant difference in the 5-year survival rates of matched sibling donor recipients [58%; 95% confidence interval (CI), 48% to 68%] and unrelated donor recipients (53%; 95% CI, 39% to 67%; P = 0.4). Among patients who underwent transplantation within 1 year after diagnosis, the 5-year survival rate of matched sibling donor recipients (76%; 95% CI, 65% to 87%) was not significantly different (P = 0.5) from that of unrelated donor recipients (70%; 95% CI, 52% to 88%). In multiple regression analysis, longer time from diagnosis to transplantation, T-cell depletion, and grades III or IV graft versus host disease were independently associated with poorer survival. Transplantation of unrelated donor bone marrow was not associated with mortality (relative risk, 1.1; 95% CI, 0.6 to 2.1; P = 0.7). CONCLUSIONS: Transplantation of bone marrow from a matched sibling donor or an HLA-A,B/DRB1-matched unrelated donor produces equivalent outcomes in patients with chronic myelogenous leukemia, particularly if the transplant takes place within 1 year after diagnosis.

Regulation of hematopoiesis through adhesion receptors.

Normal steady-state hematopoiesis takes place in the bone marrow microenvironment. Soluble factors as well as contact interactions between the hematopoietic cells and the marrow microenvironment dictate the fate of hematopoietic stem cells and progenitors. Over the last decade it has become clear that cell-cell and cell-extracellular matrix interactions through adhesion receptors play a major role in the hematopoietic process. They are required for the residence of stem cells and progenitors in the marrow, as well as for homing of stem and progenitor cells to the marrow in the setting of stem cell transplantation. Furthermore, adhesion receptors play an important role in regulation of cell behavior, either through direct activation of signal pathways important for cell survival, cell growth, and cell fate decision-making processes, or by modulating responses to growth factors. Insights in the abnormalities seen in these interactions in diseases of the hematopoietic system will help to develop better therapeutic strategies based on the pathogenesis of these diseases.

Current status of cord blood banking and transplantation in the United States and Europe.

Cord blood (CB) transplantation has expanded the ability of the transplantation community to meet the growing needs of their patients. Clinical data over the last decade show promising results in CB transplantation using blood from related as well as unrelated donors. Basic science continues to look for ways to expand the quality and quantity of CB. CB banks are now established around the world, with major efforts to standardize banking to facilitate regulation, collection, processing, and distribution as a way of providing the highest-quality CB for patient use. This review article discusses the current status of CB transplantation and banking in the United States and Europe.

Matched-pair analysis of peripheral blood stem cells compared to marrow for allogeneic transplantation.

We performed a case-control analysis of 42 patients with advanced leukemia or MDS comparing peripheral blood stem cell (PBSC) with marrow grafts (BMT) from HLA-matched sibling donors. PBSC were mobilized with G-CSF (7.5 microg/kg/day) and yielded a median of 6.7 x 10(6) CD34+ cells/kg (range, 1.6-15.0) and 2.7 x 10(8) CD3+ cells/kg (range, 1.1-7.1) vs marrow grafts with a median of 2.0 x 10(8) nucleated cells/kg (range, 1.8-2.2). Recovery was significantly faster after PBSCT compared to BMT, with a median of 17 (range, 12-26) vs 26 (range, 16-36) days, respectively, to neutrophils >0.5 x 10(9)/l (P < 0.01), and 22 (range, 12->60) vs 42 (range, 18->60) days, for platelet recovery (P < 0.01). Transplantation of >/=7 x 10(6) CD34+ cells/kg accelerated recovery to >20 x 10(9) l platelets; median 17 days (range, 12-19) vs 23 days (range, 17-36) for those receiving <7 x 10(6)/kg (P = 0.01). PBSC and marrow recipients had similar risks of grades II-IV or III-IV acute GVHD or extensive chronic GVHD (all P > 0.3). At 1 year after PBSCT and BMT, the risk of relapse was 41% and 32%, respectively (P = 0.47), and the probability of survival was 46% and 48%, respectively (P = 0.70). HLA-matched sibling PBSCT resulted in faster neutrophil and platelet engraftment compared to BMT, with no subsequent differences in acute or chronic GVHD, relapse or survival. A minimum of 7 x 10(6) CD34+ cells/kg in PBSC grafts may be required for very rapid platelet engraftment. Bone Marrow Transplantation (2000) 26, 723-728.