Umberto Galderisi

From the laboratory bench to the patient's bedside: An update on clinical trials with mesenchymal stem cells

Mesenchymal Stem Cells (MSCs) are non‐hematopoietic multi‐potent stem‐like cells that are capable of differentiating into both mesenchymal and non‐mesenchymal lineages. In fact, in addition to bone, cartilage, fat, and myoblasts, it has been demonstrated that MSCs are capable of differentiating into neurons and astrocytes in vitro and in vivo. MSCs are of interest because they are isolated from a small aspirate of bone marrow and can be easily expanded in vitro. As such, these cells are currently being tested for their potential use in cell and gene therapy for a number of human diseases. Nevertheless, there are still some open questions about origin, multipotentiality, and anatomical localization of MSCs. In this review, we discuss clinical trials based on the use of MSCs in cardiovascular diseases, such as treatment of acute myocardial infarction, endstage ischemic heart disease, or prevention of vascular restenosis through stem cell‐mediated injury repair. We analyze data from clinical trials for treatment of osteogenesis imperfecta (OI), which is a genetic disease characterized by production of defective type I collagen. We describe progress for neurological disease treatment with MSC transplants. We discuss data on amyotrophic lateral sclerosis (ALS) and on lysosomal storage diseases (Hurler syndrome and metachromatic leukodystrophy). A section of review is dedicated to ongoing clinical trials, involving MSCs in treatment of steroid refractory Graft Versus Host Disease (GVHD); periodontitis, which is a chronic disease affecting periodontium and causing destruction of attachment apparatus, heart failure, and bone fractures. Finally, we will provide information about biotech companies developing MSC therapy. J. Cell. Physiol. 211: 27–35, 2007.

Detection and Characterization of CD133 + Cancer Stem Cells in Human Solid Tumours

Background Osteosarcoma is the most common primary tumour of bone. Solid tumours are made of heterogeneous cell populations, which display different goals and roles in tumour economy. A rather small cell subset can hold or acquire stem potentials, gaining aggressiveness and increasing expectancy of recurrence. The CD133 antigen is a pentaspan membrane glycoprotein, which has been proposed as a cancer stem cell marker, since it has been previously demonstrated to be capable of identifying a cancer initiating subpopulation in brain, colon, melanoma and other solid tumours. Therefore, our aim was to observe the possible presence of cells expressing the CD133 antigen within solid tumour cell lines of osteosarcoma and, then, understand their biological characteristics and performances. Methodology and Principal Findings In this study, using SAOS2, MG63 and U2OS, three human sarcoma cell lines isolated from young Caucasian subjects, we were able to identify and characterize, among them, CD133+ cells showing the following features: high proliferation rate, cell cycle detection in a G2\M phase, positivity for Ki-67, and expression of ABCG2 transporters. In addition, at the FACS, we were able to observe the CD133+ cell fraction showing side population profile and forming sphere-clusters in serum-free medium with a high clonogenic efficiency. Conclusions Taken together, our findings lead to the thought that we can assume that we have identified, for the first time, CD133+ cells within osteosarcoma cell lines, showing many features of cancer stem cells. This can be of rather interest in order to design new therapies against the bone cancer.

Clinical Trials With Mesenchymal Stem Cells: An Update.

In the last year, the promising features of mesenchymal stem cells (MSCs), including their regenerative properties and ability to differentiate into diverse cell lineages, have generated great interest among researchers whose work has offered intriguing perspectives on cell-based therapies for various diseases. Currently the most commonly used adult stem cells in regenerative medicine, MSCs, can be isolated from several tissues, exhibit a strong capacity for replication in vitro, and can differentiate into osteoblasts, chondrocytes, and adipocytes. However, heterogeneous procedures for isolating and cultivating MSCs among laboratories have prompted the International Society for Cellular Therapy (ISCT) to issue criteria for identifying unique populations of these cells. Consequently, the isolation of MSCs according to ISCT criteria has produced heterogeneous, nonclonal cultures of stromal cells containing stem cells with different multipotent properties, committed progenitors, and differentiated cells. Though the nature and functions of MSCs remain unclear, nonclonal stromal cultures obtained from bone marrow and other tissues currently serve as sources of putative MSCs for therapeutic purposes, and several findings underscore their effectiveness in treating different diseases. To date, 493 MSC-based clinical trials, either complete or ongoing, appear in the database of the US National Institutes of Health. In the present article, we provide a comprehensive review of MSC-based clinical trials conducted worldwide that scrutinizes biological properties of MSCs, elucidates recent clinical findings and clinical trial phases of investigation, highlights therapeutic effects of MSCs, and identifies principal criticisms of the use of these cells. In particular, we analyze clinical trials using MSCs for representative diseases, including hematological disease, graft-versus-host disease, organ transplantation, diabetes, inflammatory diseases, and diseases in the liver, kidney, and lung, as well as cardiovascular, bone and cartilage, neurological, and autoimmune diseases.

Antisense oligonucleotides as therapeutic agents

Antisense oligonucleotides can block the expression of specific target genes involved in the development of human diseases. Therapeutic applications of antisense techniques are currently under investigation in many different fields. The use of antisense molecules to modify gene expression is variable in its efficacy and reliability, raising objections about their use as therapeutic agents. However, preliminary results of several clinical studies demonstrated the safety and to some extent the efficacy of antisense oligodeoxynucleotides (ODNs) in patients with malignant diseases. Clinical response was observed in some patients suffering from ovarian cancer who were treated with antisense targeted against the gene encoding for the protein kinase C‐alpha. Some hematological diseases treated with antisense oligos targeted against the bcr/abl and the bcl2 mRNAs have shown promising clinical response. Antisense therapy has been useful in the treatment of cardiovascular disorders such as restenosis after angioplasty, vascular bypass graft occlusion, and transplant coronary vasculopathy. Antisense oligonucleotides also have shown promise as antiviral agents. Several investigators are performing trials with oligonucleotides targeted against the human immunodeficiency virus‐1 (HIV‐1) and hepatitis viruses. Phosphorothioate ODNs now have reached phase I and II in clinical trials for the treatment of cancer and viral infections, so far demonstrating an acceptable safety and pharmacokinetic profile for continuing their development. The new drug Vitravene, based on a phosphorothioate oligonucleotide designed to inhibit the human cytomegalovirus (CMV), promises that some substantial successes can be reached with the antisense technique. J. Cell. Physiol. 181:251–257, 1999.

Cell cycle regulation and neural differentiation

The general mechanisms that control the cell cycle in mammalian cells have been studied in depth and several proteins that are involved in the tight regulation of cell cycle progression have been identified. However, the analysis of which molecules participate in cell cycle exit of specific cell lineages is not exhaustive yet. Moreover, the strict relation between cell cycle exit and induction of differentiation has not been fully understood and seems to depend on the cell type. Several in vivo and in vitro studies have been performed in the last few years to address these issues in cells of the nervous system. In this review, we focus our attention on cyclin–cyclin-dependent kinase complexes, cyclin kinase inhibitors, genes of the retinoblastoma family, p53 and N-Myc, and we aim to summarize the latest evidence indicating their involvement in the control of the cell cycle and induction of differentiation in different cell types of the peripheral and central nervous systems. Studies on nervous system tumors and a possible contributory role in tumorigenesis of polyomavirus T antigen are reported to point out the critical contribution of some cell cycle regulators to normal neural and glial development.

Molecular pathways involved in neural in vitro differentiation of marrow stromal stem cells

In recent years several reports have claimed to demonstrate trans‐differentiation, namely that stem cells have been derived from a given tissue and have differentiated into phenotypes characteristic of different tissues following transplantation or in vitro treatment. For example, the mesenchymal stem cells, also referred to as marrow stromal stem cells (MSCs), present in bone marrow, have been induced to differentiate into neurons. We decided to investigate this phenomenon more in depth by a molecular and morphological follow‐up. We analyzed the biochemical pathways that are currently induced to trigger neuron‐like commitment and maturation of MSCs. Our studies suggest that: (i) the increase in cAMP, induced to differentiate MSCs, activates the classical PKA pathway and not through the exchange protein directly activated by cAMP (EPAC), a guanine nucleotide exchange factor for the small GTPase Rap1 and Rap2; (ii) MEK–ERK signaling could contribute to neural commitment and differentiation; (iii) CaM KII activity seems dispensable for neuron differentiation. On the contrary, its inhibition could contribute to rescuing differentiating cells from death. Our research also indicates that the currently used in vitro differentiation protocols, while they allow the early steps of neural differentiation to take place, are not able to further sustain this process.

Differentiation and apoptosis of neuroblastoma cells: role of N-myc gene product.

To clarify the role and function of the N‐myc product in cell differentiation and apoptosis, we used the antisense oligonucleotide technique to inhibit N‐myc gene expression in neuroblastoma cells with different phenotypes: intermediate (I) and neuronal (N), or Schwann‐glia (S), respectively. The results suggest that N‐myc operates along different pathways. Inhibiting N‐myc gene expression either results in suppression of cell proliferation or in induction of differentiation and/or apoptosis. J. Cell. Biochem. 73:97–105, 1999.

Blockade of glutamate mGlu5 receptors in a rat model of neuropathic pain prevents early over-expression of pro-apoptotic genes and morphological changes in dorsal horn lamina II.

We used rats with a sciatic nerve chronic constrictive injury (CCI) and combined behavioural, molecular and morphological approaches to assess the involvement of mGlu5 receptors in neuropathic pain-associated hyperalgesia and spinal cord neuron apoptosis. Mechanical and thermal hyperalgesia developed 2-3 days after surgery. Morphological changes in the ipsilateral L4-L5 lamina II consisted of: (i) cell loss (38 +/- 5%), (ii) increased TUNEL-positive profiles, (iii) decreased SP-immunoreactive primary afferents, and (iv) reactive gliosis. Molecular expression data suggested a bi-phasic response of bcl-2 family genes in CCI. An early (2-3 days post-CCI) E2F1- and p53-independent apoptosis appeared in the spinal cord as the pro-apoptotic bax gene increased (320 +/- 19%), followed by an increased expression of the anti-apoptotic bcl-2 and bcl-xL genes (60 +/- 11% and 110 +/- 15%, respectively) 7 days from CCI. The selective mGlu5 receptor antagonist, MPEP (2 mg/kg i.p. twice daily), prevented the development of thermal hyperalgesia and transiently reduced mechanical hyperalgesia. Despite the MPEP treatment, which normalised bax/bcl-2 and bcl-xL/bcl-xS ratios at all times post-CCI, mechanical hyperalgesia reappeared by 7 days after CCI. Similarly, MPEP was cytoprotective at 3, but not 7 days post-CCI. This study shows that: (a) spinal cord neuron loss may be triggered by a p53- and E2F1-independent apoptosis in lamina II with the participation of glutamate mGlu5 receptors, (b) these receptors seem to be involved transiently, as their blockade was no longer protective by 7 days CCI, and (c) this delayed cell death occurred in the absence of Bax activation, suggesting the involvement of an alternative death pathway.

The retinoblastoma gene is involved in multiple aspects of stem cell biology

Genetic programs controlling self-renewal and multipotentiality of stem cells have overlapping pathways with cell cycle regulation. Components of cell cycle machinery can play a key role in regulating stem cell self-renewal, proliferation, differentiation and aging. Among the negative regulators of cell cycle progression, the RB family members play a prominent role in controlling several aspects of stem cell biology. Stem cells contribute to tissue homeostasis and must have molecular mechanisms that prevent senescence and hold ‘stemness’. RB can induce senescence-associated changes in gene expression and its activity is downregulated in stem cells to preserve self-renewal. Several reports evidenced that RB could play a role in lineage specification of several types of stem cells. RB has a role in myogenesis as well as in cardiogenesis. These effects are not only related to its role in suppressing E2F-responsive genes but also to its ability to modulate the activity of tissue-specific transcription factors. RB is also involved in adipogenesis through a strict control of lineage commitment and differentiation of adipocytes as well in determining the switch between brown and white adipocytes. Also, hematopoietic progenitor cells utilize the RB pathway to modulate cell commitment and differentiation. In this review, we will also discuss the role of the other two RB family members: Rb2/p130 and p107 showing that they have both specific and overlapping functions with RB gene.

Insulin-like growth factor binding proteins 4 and 7 released by senescent cells promote premature senescence in mesenchymal stem cells

Cellular senescence is the permanent arrest of cell cycle, physiologically related to aging and aging-associated diseases. Senescence is also recognized as a mechanism for limiting the regenerative potential of stem cells and to protect cells from cancer development. The senescence program is realized through autocrine/paracrine pathways based on the activation of a peculiar senescence-associated secretory phenotype (SASP). We show here that conditioned media (CM) of senescent mesenchymal stem cells (MSCs) contain a set of secreted factors that are able to induce a full senescence response in young cells. To delineate a hallmark of stem cells SASP, we have characterized the factors secreted by senescent MSC identifying insulin-like growth factor binding proteins 4 and 7 (IGFBP4 and IGFBP7) as key components needed for triggering senescence in young MSC. The pro-senescent effects of IGFBP4 and IGFBP7 are reversed by single or simultaneous immunodepletion of either proteins from senescent-CM. The blocking of IGFBP4/7 also reduces apoptosis and promotes cell growth, suggesting that they may have a pleiotropic effect on MSC biology. Furthermore, the simultaneous addition of rIGFBP4/7 increased senescence and induced apoptosis in young MSC. Collectively, these results suggest the occurrence of novel-secreted factors regulating MSC cellular senescence of potential importance for regenerative medicine and cancer therapy.