Tiziano Barberi - Academia.edu (original) (raw)

Papers by Tiziano Barberi

European journal of histochemistry : EJH, 1997

Stem Cells and Development, 2014

Humans and nonhuman primates (NHPs) are similar in size, behavior, physiology, biochemistry, stru... more Humans and nonhuman primates (NHPs) are similar in size, behavior, physiology, biochemistry, structure and function of organs, and complexity of the immune system. Research on NHPs generates complementary data that bridge translational research from small animal models to humans. NHP models of human disease offer unique opportunities to develop stem cell-based therapeutic interventions that directly address relevant and challenging translational aspects of cell transplantation therapy. These include the use of autologous induced pluripotent stem cell-derived cellular products, issues related to the immune response in autologous and allogeneic setting, pros and cons of delivery techniques in a clinical setting, as well as the safety and efficacy of candidate cell lines. The NHP model allows the assessment of complex physiological, biochemical, behavioral, and imaging end points, with direct relevance to human conditions. At the same time, the value of using primates in scientific research must be carefully evaluated and timed due to expense and the necessity for specialized equipment and highly trained personnel. Often it is more efficient and useful to perform initial proof-of-concept studies for new therapeutics in rodents and/or other species before the pivotal studies in NHPs that may eventually lead to first-in-human trials. In this report, we present how the Southwest National Primate Research Center, one of seven NIH-funded National Primate Research Centers, may help the global community in translating promising technologies to the clinical arena.

Methods in enzymology, 2006

Human embryonic stem cells (hESC) provide a potentially unlimited source of specialized cell type... more Human embryonic stem cells (hESC) provide a potentially unlimited source of specialized cell types for regenerative medicine. Nonetheless, one of the key requirements used to fulfill this potential is the ability to direct the differentiation of hESC to selective fates in vitro. Studies have reported the development of culture strategies to derive multipotent mesenchymal precursors from hESCs in vitro. This chapter reviews the techniques that allow the selective derivation of such precursors and their differentiation toward various mesenchymal cell types. It also discusses current limitations and future perspectives on the use of hESC-derived mesenchymal tissues.

Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1995

Studies on the effect of retinoic acid (RA) and 1,25-dihydroxyvitamin (D3) on the differentiation... more Studies on the effect of retinoic acid (RA) and 1,25-dihydroxyvitamin (D3) on the differentiation of leukemic cells have provided insight into the cellular and molecular mechanisms underlying hematopoietic cell differentiation. We have evaluated the combined effect of these chemical inducers on the differentiation of HL-60 and AML-193 promyelocytic leukemia cell lines. Simultaneous RA+D3 addition potentiated leukemic cell maturation up to mature phagocytic cells. Interestingly, AML-193 cells induced with D3 and RA displayed a typical neutrophilic morphology while exhibiting properties specific to monocytic cells, e.g., high expression of CD14 membrane antigen, capacity to bind bacterial lipopolysaccharide, and monocytic-specific esterase activity; this hybrid granulomonocytic (GM) phenotype was not observed upon initial incubation with one inducer and later addition of the other. Parallel control studies were performed with purified normal GM progenitors, triggered by interleukin 3+...

Blood, 1995

We have evaluated the effects of the flt3 receptor ligand (FL) on hematopoietic progenitors/stem ... more We have evaluated the effects of the flt3 receptor ligand (FL) on hematopoietic progenitors/stem cells (HPCs/HSCs) stringently purified from adult peripheral blood and grown in different culture systems. In these experiments HPCs/HSCs were treated with FL +/- kit ligand (KL) +/- monocyte colony-stimulatory factor (M-CSF). In clonogenetic HPC culture supplemented with interleukin-3 (IL-3)/granulomonocyte-CSF (GM-CSF)/erythropoietin (Epo), FL potentiates colony-forming unit (CFU)-GM proliferation in terms of colony number and size, but exerts little effect on burst-forming units-erythroid (BFU-E) and CFU-granulocyte erythroid megakaryocyte macrophage (CFU-GEMM) growth, whereas KL enhances the proliferation of all HPC types; combined FL+KL +/- M-CSF treatment causes a striking shift of CFU-GM colonies from granulocytic to monocytic differentiation. In liquid suspension HPC culture, FL alone induces differentiation along the monocytic and to a minor extent the basophilic lineages, where...

Cancer research, Jan 15, 1995

Acute promyelocytic leukemia (APL) is characterized by a t(15;17) chromosomal translocation with ... more Acute promyelocytic leukemia (APL) is characterized by a t(15;17) chromosomal translocation with breakpoints within the retinoic acid alpha receptor (RAR alpha) gene on 17 and the PML gene, which encodes a putative transcription factor, on 15. A PML-RAR alpha fusion protein is formed as a consequence of the translocation. We show here that expression of the PML-RAR alpha protein in K562 erythroleukemia cells results in a reduced expression of erythroid differentiation markers and a reduced sensitivity to the erythroid differentiative action of heme. Overexpression of RAR alpha, but not of PML, elicited a similar inhibition of K562 erythroid differentiation. These findings indicate that overexpression of either RAR alpha or PML/RAR alpha interferes with erythroid differentiation and support the hypothesis that RAR alpha is involved in the regulation of normal hematopoiesis and alteration of the RAR alpha signaling by PML/RAR alpha is implicated in the promyelocytic leukemogenesis.

Cancer research, Jan 15, 1994

We have analyzed the differentiation program of a U937 promonocytic leukemia clone transduced wit... more We have analyzed the differentiation program of a U937 promonocytic leukemia clone transduced with the acute promyelocytic leukemia specific PML/RAR alpha fusion gene, the expression of which is under the control of the inducible metallothionine (MT) I promoter (MTPR9 clone). MTPR9 cells treated with Zn2+ hence exhibit levels of PML-RAR alpha protein as high as fresh acute promyelocytic leukemia blasts. In the absence of Zn2+, i.e., upon low level PML/RAR alpha expression, 1,25-dihydroxyvitamin D3 (D3) and particularly D3 plus transforming growth factor beta 1 (TGF-beta 1) induced terminal differentiation of MTPR9 cells (as observed in "wild-type" U937 cells), on the basis of morphology, membrane antigen pattern, and functional criteria. Conversely, in the presence of Zn2+, D3 and D3 plus TGF-beta 1 failed to induce terminal differentiation, as evaluated by the above parameters. Interestingly, retinoic acid (RA) treatment suppresses the differentiation blockade induced by ...

Methods in Molecular Biology, 2013

Flow Cytometry-Sorting (FCM-Sorting) is a technique commonly used to identify and isolate specifi... more Flow Cytometry-Sorting (FCM-Sorting) is a technique commonly used to identify and isolate specific types of cells from a heterogeneous population of live cells. Here we describe a multicolor flow cytometry technique that uses five distinct cell surface antigens to isolate four live populations with different surface antigen profiles. These profiles were used to help distinguishing between neural and nonneural (the lens) ectoderm derivatives within a highly heterogenous population of differentiating human embryonic stem cells (hESC).

We evaluated phenotype and apoptotic status of normal CD4 ؉ CD69 ؉ and CD8 ؉ CD69 ؉ peripheral bl... more We evaluated phenotype and apoptotic status of normal CD4 ؉ CD69 ؉ and CD8 ؉ CD69 ؉ peripheral blood T-lymphocytes after short-term challenge with escalating concentrations of phytohemagglutinin (PHA). The frequency of CD69-coexpressing CD4 ؉ and CD8 ؉ T-cells and CD69 staining intensity increased following T-cell mitogenic stimulation; these changes were proportional to PHA concentration in culture medium. A considerable fraction of lymphocytes underwent blast transformation, displaying increased forward and side scatter signals. Interestingly enough, PHA-responsive T-cells exhibited a predominantly CD25 neg CD38 neg TCR␣␤ pos phenotype; APO-1/Fas antigen (CD95) could be detected on a minority of activated CD69 ؉ T-cells. A considerable proportion of CD69 ؉ lymphocytes expressed intracellular perforin; in addition, an average 16 ؎ 6% CD69 ؉ T-lymphocytes were apoptotic after 4 h of stimulation, as evaluated by 7-amino-actinomycin-D staining and by annexin-V binding. CD69 ؉ activated lymphocytes comprise phenotypically heterogeneous cell subpopulations potentially devoted to diverse immunological functions, i.e., proliferation, apoptosis, or cell cytotoxicity; moreover, our findings indicate that CD69 expression is proportional to the intensity of the activating stimulus and that the capacity to upregulate CD69 antigen following short-term mitogenic challenge may be restricted to unactivated CD38 neg CD25 neg TCR␣␤ pos T-lymphocytes. Cytometry (Comm. Clin. Cytometry) 38:95-101, 1999.

Methods in Enzymology, 2006

European Journal of Biochemistry, 2000

We have investigated the expression of transferrin receptor (TfR) iron regulatory protein-1 (IRP-... more We have investigated the expression of transferrin receptor (TfR) iron regulatory protein-1 (IRP-1) and iron regulatory protein-2 (IRP-2) in liquid suspension culture of purified hematopoietic progenitor cells (HPCs) induced by a growth factor stimulus to proliferation and unilineage differentiation/maturation through the erythroid, granulocytic, monocytic and megakaryocytic lineages. In initial HPC differentiation, TfR expression is induced in both erythroid and granulopoietic cultures. In late HPC differentiation (i.e. starting from day 5 of culture) and then differentiated precursor maturation, the TfR gene is highly expressed in the erythroid lineage, whereas it is sharply downmodulated in the granulopoietic, monocytopoietic and megakaryocytic series. The elevated TfR expression in erythroid cells is: (a) mediated through a high rate of TfR gene transcription; (b) modulated by intracellular iron levels; (c) mediated by TfR mRNA stabilization through the iron regulatory protein (IRP), in that IRP-1 activity is high in erythroid lineage as compared to the levels observed in other hemopoietic lineages; and (d) dependent on exogenous erythropoietin (Epo) (this is indicated by the marked TfR and IRP-1/IRP-2 downmodulation after Epo starvation). Interestingly, analysis of IRP-1 and IRP-2 expression during hemopoietic differentiation showed that: (a) IRP-1 expression was maintained during all steps of erythroid differentiation, while it was lost in the other hemopoietic lineages; (b) IRP-2 expression was observed during all stages of hemopoietic differentiation in all four lineages. However, IRP-1 and IRP-2 expression and activity are induced when monocytes, which express only low levels of IRP-1 and IRP-2, are induced to maturation to macrophages. These studies indicate that: (a) in normal erythropoiesis, the hyperexpression of TfR, starting from early erythroid HPC differentiation, is Epo-dependent and mediated via transcriptional and post-transcriptional mechanisms; (b) in the granulopoietic, monocytopoietic and megakaryocytic pathways, the TfR is first induced and then downmodulated (the latter phenomenon is mediated via transcriptional suppression of the TfR gene and IRP inactivation).

Stem Cells and Development, 2010

Recent findings emphasized a critical role for the Wnt signaling pathway during the early steps o... more Recent findings emphasized a critical role for the Wnt signaling pathway during the early steps of embryogenesis, including the development of the hematopoietic system and cardiac development. To date, the role of Wnt in promoting or inhibiting development of both tissues was discussed controversially, dependent on species and time point of expression. Differentiation of embryonic stem cells (ESC) recapitulates early stages of mammalian development. In the present study, we generated murine ESC lines overexpressing Wnt1 (Wnt1 ES). When induced to differentiate toward the cardiomyocytic lineage, Wnt1 ES showed a significant increased ability to generate cardiomyocytes when compared with a control ESC (control ES) line. In addition, Wnt1 ES cells were unable to form hematopoietic cells, whereas development of endothelial cells, a cell type closely associated with blood during embryogenesis, was comparable to control ES. Finally, cardiac differentiation was markedly decreased by the addition of the Wnt antagonist Dkk-1 to the culture medium. These findings suggest that Wnt1 may regulate differentiation of immature mesodermal cells in a tissue-specific manner.

Proceedings of the National Academy of Sciences, 1995

The retinoblastoma (RB) gene specifies a nuclear phosphoprotein (pRb 105), which is a prototype t... more The retinoblastoma (RB) gene specifies a nuclear phosphoprotein (pRb 105), which is a prototype tumor suppressor inactivated in a variety of human tumors. Recent studies suggest that RB is also involved in embryonic development of murine central nervous and hematopoietic systems. We have investigated RB expression and function in human adult hematopoiesis-i.e., in liquid suspension culture of purified quiescent hematopoietic progenitor cells (HPCs) induced by growth factor stimulus to proliferation and unilineage differentiation/maturation through the erythroid or Proc. Natl. Acad ScL USA 92 (1995)

Proceedings of the National Academy of Sciences, 2004

Human embryonic stem (hES) cells are defined by their extensive self-renewal capacity and their p... more Human embryonic stem (hES) cells are defined by their extensive self-renewal capacity and their potential to differentiate into any cell type of the human body. The challenge in using hES cells for developmental biology and regenerative medicine has been to direct the wide differentiation potential toward the derivation of a specific cell fate. Within the nervous system, hES cells have been shown to differentiate in vitro into neural progenitor cells, neurons, and astrocytes. However, to our knowledge, the selective derivation of any given neuron subtype has not yet been demonstrated. Here, we describe conditions to direct hES cells into neurons of midbrain dopaminergic identity. Neuroectodermal differentiation was triggered on stromal feeder cells followed by regional specification by means of the sequential application of defined patterning molecules that direct in vivo midbrain development. Progression toward a midbrain dopamine (DA) neuron fate was monitored by the sequential expression of key transcription factors, including Pax2, Pax5, and engrailed-1 (En1), measurements of DA release, the presence of tetrodotoxin-sensitive action potentials, and the electron-microscopic visualization of tyrosinehydroxylase-positive synaptic terminals. High-yield DA neuron derivation was confirmed from three independent hES and two monkey embryonic stem cell lines. The availability of unlimited numbers of midbrain DA neurons is a first step toward exploring the potential of hES cells in preclinical models of Parkinson's disease. This experimental system also provides a powerful tool to probe the molecular mechanisms that control the development and function of human midbrain DA neurons.

PLoS Medicine, 2005

Human embryonic stem cells provide access to the earliest stages of human development and may ser... more Human embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. Thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue-restricted precursors. Here, we present culture conditions for the derivation of unlimited numbers of pure mesenchymal precursors from human embryonic stem cells and demonstrate multilineage differentiation into fat, cartilage, bone, and skeletal muscle cells. Our findings will help to elucidate the mechanism of mesoderm specification during embryonic stem cell differentiation and provide a platform to efficiently generate specialized human mesenchymal cell types for future clinical applications.

Nature Medicine, 2007

Human embryonic stem cells (hESCs) are a promising source for cell therapy in degenerative diseas... more Human embryonic stem cells (hESCs) are a promising source for cell therapy in degenerative diseases. A key step in establishing the medical potential of hESCs is the development of techniques for the conversion of hESCs into tissue-restricted precursors suitable for transplantation. We recently described the derivation of multipotent mesenchymal precursors from hESCs. Nevertheless, our previous study was limited by the requirement for mouse feeders and the lack of in vivo data. Here we report a stroma-free induction system for deriving mesenchymal precursors. Selective culture conditions and fluorescence-activated cell sorting (FACS)-mediated purification yielded multipotent mesenchymal precursors and skeletal myoblasts. Skeletal muscle cells undergo in vitro maturation resulting in myotube formation and spontaneous twitching. We found that hESC-derived skeletal myoblasts were viable after transplantation into the tibialis anterior muscle of SCID/Beige mice, as assessed by bioluminescence imaging. Lack of teratoma formation and evidence of long-term myoblast engraftment suggests considerable potential for future therapeutic applications.

Nature Biotechnology, 2003

culture, yield variable differentiation results or are limited to the generation of selected neur... more culture, yield variable differentiation results or are limited to the generation of selected neural subtypes. Here we provide a set of coculture conditions that allows rapid and efficient derivation of most central nervous system phenotypes. The fate of both fertilizationand nuclear transfer-derived ES (ntES) cells was directed selectively into neural stem cells, astrocytes, oligodendrocytes or neurons. Specific differentiation into γ-aminobutyric acid (GABA), dopamine, serotonin or motor neurons was achieved by defining conditions to induce forebrain, midbrain, hindbrain and spinal cord identity. Neuronal function of ES cell-derived dopaminergic neurons was shown in vitro by electron microscopy, measurement of neurotransmitter release and intracellular recording. Furthermore, transplantation of ES and ntES cell-derived dopaminergic neurons corrected the phenotype of a mouse model of Parkinson disease, demonstrating an in vivo application of therapeutic cloning in neural disease.

Nature Biotechnology, 2008

Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although a... more Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest-related disorders.

Nature Biotechnology, 2007

Medical Hypotheses, 1998

Both human cell lines HL-60 and AML-193 exhibit a myeloblastic and promyelocytic morphology, resp... more Both human cell lines HL-60 and AML-193 exhibit a myeloblastic and promyelocytic morphology, respectively, but may be regarded as bipotent leukemic precursors. They can be triggered to differentiate to either granulocytes or monocytes upon retinoic acid (RA) or 1,25-dihydroxyvitamin D (D3) addition, respectively. We have investigated the effect of combined addition of these chemical inducers on the in-vitro differentiation of both cell lines. RA and D3 added together exert synergistic effects on the in-vitro maturation of these myeloid cell lines. Interestingly, the additive effects were lost if the cells were incubated with the inducers added at sequential times. The synergistic effect could be transposed in vivo and could be clinically significant in the treatment of the promyelocytic leukemia. This clinical strategy may help to prevent retinoic acid resistance or to overcome it in patients relapsed after RA therapy and usually unresponsive to a reinduction therapy with RA alone.