Physical characterization of hematopoietic stem cells using multidirectional label-free light scatterings.

An experimental setup capable of measuring simultaneous 2D scattered light angular distribution from two directions to study cell morphology without the use of bio-labels was developed. Experiments with hematopoietic stem cells (CD34 cells) show good agreement with detailed numerical simulations of light scattering. Numerical simulations and computer models of cells are used to identify physical features of cells with the largest scattering cross sections.

Novel evidence that pituitary gonadotropins directly stimulate human leukemic cells-studies of myeloid cell lines and primary patient AML and CML cells.

We recently reported that normal hematopoietic stem cells express functional pituitary sex hormone (SexH) receptors. Here we report for the first time that pituitary-secreted gonadotrophins stimulate migration, adhesion, and proliferation of several human myeloid and lymphoid leukemia cell lines. Similar effects were observed after stimulation of human leukemic cell lines by gonadal SexHs.

Mesenchymal stromal cells derived from various tissues: Biological, clinical and cryopreservation aspects.

Originally isolated from bone marrow, mesenchymal stromal cells (MSCs) have since been obtained from various fetal and post-natal tissues and are the focus of an increasing number of clinical trials. Because of their tremendous potential for cellular therapy, regenerative medicine and tissue engineering, it is desirable to cryopreserve and bank MSCs to increase their access and availability. A remarkable amount of research and resources have been expended towards optimizing the protocols, freezing media composition, cooling devices and storage containers, as well as developing good manufacturing practices in order to ensure that MSCs retain their therapeutic characteristics following cryopreservation and that they are safe for clinical use.

Further evidence that paroxysmal nocturnal haemoglobinuria is a disorder of defective cell membrane lipid rafts.

The glycolipid glycosylphosphatidylinositol anchor (GPI-A) plays an important role in lipid raft formation, which is required for proper expression on the cell surface of two inhibitors of the complement cascade, CD55 and CD59. The absence of these markers from the surface of blood cells, including erythrocytes, makes the cells susceptible to complement lysis, as seen in patients suffering from paroxysmal nocturnal haemoglobinuria (PNH). However, the explanation for why PNH-affected hematopoietic stem/progenitor cells (HSPCs) expand over time in BM is still unclear.

Progress in RNAi-mediated Molecular Therapy of Acute and Chronic Myeloid Leukemia.

Leukemias arise from genetic alterations in normal hematopoietic stem or progenitor cells, leading to impaired regulation of proliferation, differentiation, apoptosis, and survival of the transformed cells. With the advent of RNA interference (RNAi) and the short interfering RNA (siRNA) as its pharmacological mediator, it is becoming possible to modulate specific targets at will. This article summarizes current attempts to utilize RNAi reagents for therapy of leukemias, focusing on acute and chronic myeloid leukemia.

Polymeric nanoparticle-mediated silencing of CD44 receptor in CD34+ acute myeloid leukemia cells.

The adhesion receptor CD44 plays an important role in the survival and retention of leukemic stem/progenitor cells (LSPC) within the bone marrow (BM) niche, as well as in the high relapse rates of acute myeloid leukemia (AML). Down-regulating CD44 could be clinically relevant not only for suppression of the deregulated function of LSPC but also in LSPC response to chemotherapeutic agents. Small interfering RNA (siRNA) delivery is a promising approach for AML treatment, and we recently reported effective siRNA delivery into difficult-to-transfect AML cell lines using lipid-substituted polyethylenimine/siRNA complexes (polymeric nanoparticles).

Sustained expression of coagulation factor IX by modified cord blood-derived mesenchymal stromal cells.

Background: Hemophilia B patients are subject to frequent and spontaneous bleeding caused by a deficiency of clotting factor IX (FIX). Mesenchymal stromal cells (MSCs) have been used in cellular therapies as a result of their immunomodulatory properties, the ability to home to sites of injury and their amenability to various ex vivo modifications, including lentiviral-mediated gene transfer.

Methods: MSCs were isolated from human umbilical cord blood and differentiated into adipogenic, chondrogenic and osteogenic lineages.

Migration, proliferation, and differentiation of cord blood mesenchymal stromal cells treated with histone deacetylase inhibitor valproic Acid.

Mesenchymal stromal cells (MSC) have great potential for cellular therapies as they can be directed to differentiate into certain lineages or to exert paracrine effects at sites of injury. The interactions between stromal cell-derived factor (SDF)-1 and its receptors CXCR4 and CXCR7 play pivotal roles in the migration of MSC to injured tissues. We evaluated whether a histone deacetylase inhibitor valproic acid (VPA) modulates the migration of cord blood (CB-) derived MSC towards SDF-1 and their proliferation and differentiation.

Fibronectin-Alginate microcapsules improve cell viability and protein secretion of encapsulated Factor IX-engineered human mesenchymal stromal cells.

Continuous delivery of proteins by engineered cells encapsu-lated in biocompatible polymeric microcapsules is of considerable therapeutic potential. However, this technology has not lived up to expectations due to inadequate cell--matrix interactions and subsequent cell death. In this study we hypoth-esize that the presence of fibronectin in an alginate matrix may enhance the viability and functionality of encapsulated human cord blood-derived mesenchymal stromal cells (MSCs) expressing the human Factor IX (FIX) gene.

Enhancing the migration ability of mesenchymal stromal cells by targeting the SDF-1/CXCR4 axis.

Mesenchymal stromal cells (MSCs) are currently being investigated in numerous clinical trials of tissue repair and various immunological disorders based on their ability to secrete trophic factors and to modulate inflammatory responses. MSCs have been shown to migrate to sites of injury and inflammation in response to soluble mediators including the chemokine stromal cell-derived factor-(SDF-)1, but during in vitro culture expansion MSCs lose surface expression of key homing receptors particularly of the SDF-1 receptor, CXCR4. Here we review studies on enhancement of SDF-1-directed migration of MSCs with the premise that their improved recruitment could translate to therapeutic benefits.

Cell-matrix Interactions of Factor IX (FIX)-engineered human mesenchymal stromal cells encapsulated in RGD-alginate vs. fibrinogen-alginate microcapsules.

The success of cell microencapsulation technology in tissue engineering and protein delivery applications depends on the viability and functionality of the encapsulated cells, which in turn are dependent upon cell/matrix interactions. In this work, we compared the viability of cord blood-derived mesenchymal stromal cells (CB MSCs), engineered to secrete factor IX (FIX) for hemophilia treatment, and encapsulated in arginine-glycine-aspartate (RGD)-alginate versus fibrinogen-alginate microcapsules. We evaluated the effect of the biomimetic matrix on cell attachment, proliferation, and secretion of FIX.

CXCR4 transfection of cord blood mesenchymal stromal cells with the use of cationic liposome enhances their migration toward stromal cell-derived factor-1.

Background Aims: The interaction between stromal cell-derived factor (SDF)-1 and its receptor CXCR4 is one of the mechanisms by which mesenchymal stromal cells (MSCs) are recruited to sites of injury. SDF-1 is upregulated in damaged tissues, but because the surface expression of CXCR4 on cultured MSCs is low, we investigated whether the delivery of CXCR4 into MSCs with the use of the cationic liposomal reagent IBAfect would increase their migration toward SDF-1.

Methods: We examined (i) the effect of MSC confluency, passage number, duration of transfection and amount of IBAfect and plasmid on transfection efficiency as determined by flow cytometric analysis of CXCR4 and (ii) whether IBAfect-mediated CXCR4 transfection affected the viability, proliferation and differentiation of MSCs as well as their response toward an SDF-1 gradient in a trans-Matrigel migration assay.

Innate immunity as orchestrator of bone marrow homing for hematopoietic stem/progenitor cells.

The first step that precedes hematopoietic transplantation is elimination of pathological hematopoiesis by administration of myeloablative doses of radiochemotherapy. This eliminates hematolymphopoietic cells and at the same time damages hematopoietic microenvironment in bone marrow (BM). The damage of BM tissue leads to activation of complement cascade (CC), and bioactive CC cleavage fragments modulate several steps of BM recovery after transplantation of hematopoietic stem progenitor cells (HSPCs).

Encapsulation of factor IX-engineered mesenchymal stem cells in fibrinogen-alginate microcapsules enhances their viability and transgene secretion.

Cell microencapsulation holds significant promise as a strategy for cellular therapies; however, inadequate survival and functionality of the enclosed cells limit its application in hemophilia treatment. Here, we evaluated the use of alginate-based microcapsules to enhance the viability and transgene secretion of human cord blood-derived mesenchymal stem cells in three-dimensional cultures. Given the positive effects of extracellular matrix molecules on mesenchymal stem cell growth, we tested whether fibrinogen-supplemented alginate microcapsules can improve the efficiency of encapsulated factor IX-engineered mesenchymal stem cells as a treatment of hemophilia B.

Low-intensity pulsed ultrasound-mediated stimulation of hematopoietic stem/progenitor cell viability, proliferation and differentiation in vitro.

Low-intensity pulsed ultrasound (LIPUS) stimulated the viability, proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) from fresh and cryopreserved peripheral blood leukapheresis product, as well as cord blood when applied for 10 min each day for 4 days. Cell viability, proliferation and differentiation were assessed on day 5 by viable cell counting, MTS proliferation assay, flow cytometry, and colony-forming unit assay. LIPUS stimulation: (i) enhanced the proliferation of fresh HSPC and maintained the viability of cryopreserved HSPC in vitro; (ii) did not affect the percentage of CD34(+) and CD14(+) cells; and (iii) enhanced burst-forming unit-erythroid colony formation.

The expanding family of bone marrow homing factors for hematopoietic stem cells: stromal derived factor 1 is not the only player in the game.

The α-chemokine stromal derived factor 1 (SDF-1), which binds to the CXCR4 and CXCR7 receptors, directs migration and homing of CXCR4+ hematopoietic stem/progenitor cells (HSPCs) to bone marrow (BM) and plays a crucial role in retention of these cells in stem cell niches. However, this unique role of SDF-1 has been recently challenged by several observations supporting SDF-1-CXCR4-independent BM homing. Specifically, it has been demonstrated that HSPCs respond robustly to some bioactive lipids, such as sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), and migrate in response to gradients of certain extracellular nucleotides, including uridine triphosphate (UTP) and adenosine triphosphate (ATP).

Hematopoietic Stem Cell Mobilization and Homing after Transplantation: The Role of MMP-2, MMP-9, and MT1-MMP.

Hematopoietic stem/progenitor cells (HSPCs) are used in clinical transplantation to restore hematopoietic function. Here we review the role of the soluble matrix metalloproteinases MMP-2 and MMP-9, and membrane type (MT)1-MMP in modulating processes critical to successful transplantation of HSPC, such as mobilization and homing. Growth factors and cytokines which are employed as mobilizing agents upregulate MMP-2 and MMP-9.

Mesenchymal stromal cells derived from umbilical cord blood migrate in response to complement C1q.

Background Aims: Mesenchymal stromal cells (MSC) have great potential for tissue regeneration and cellular therapy. They migrate preferentially to sites of inflammation and tissue injury, but the molecular signals that guide them to their target tissue remain to be elucidated. We have shown that complement component 1 subcomponent q (C1q) enhances the homing-related response of hematopoietic stem/progenitor cells.

Membrane Type-1 Matrix Metalloproteinase Expression in Acute Myeloid Leukemia and Its Upregulation by Tumor Necrosis Factor-α.

Membrane type-1 matrix metalloproteinase (MT1-MMP) has been implicated in tumor invasion, as well as trafficking of normal hematopoietic cells, and acts as a physiologic activator of proMMP-2. In this study we examined MT1-MMP expression in primary acute myeloid leukemia (AML) cells. Because tumor necrosis factor (TNF)-α is known to be elevated in AML, we also investigated the effect of TNF-α on MT1-MMP expression.

Cationic liposome-mediated CXCR4 gene delivery into hematopoietic stem/progenitor cells: implications for clinical transplantation and gene therapy.

The chemokine stromal cell-derived factor (SDF)-1α/CXCL12 and its receptor CXC chemokine receptor 4 (CXCR4) play a crucial role in the homing/engraftment and retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow. It has been shown using the viral gene transfer technique that CXCR4 overexpression on human CD34(+) HSPC significantly improves their engraftment in murine models. However, clinical trials with gene therapy have revealed safety concerns related to the immunogenicity of the viral carriers, due to the random integration of viral genes into the host genome.

Label-free and noninvasive optical detection of the distribution of nanometer-size mitochondria in single cells.

A microfluidic flow cytometric technique capable of obtaining information on nanometer-sized organelles in single cells in a label-free, noninvasive optical manner was developed. Experimental two-dimensional (2D) light scattering patterns from malignant lymphoid cells (Jurkat cell line) and normal hematopoietic stem cells (cord blood CD34+ cells) were compared with those obtained from finite-difference time-domain simulations. In the simulations, we assumed that the mitochondria were randomly distributed throughout a Jurkat cell, and aggregated in a CD34+ cell.

Microscope-based label-free microfluidic cytometry.

A microscope-based label-free microfluidic cytometer capable of acquiring two dimensional light scatter patterns from single cells, pattern analysis of which determines cellular information such as cell size, orientation and inner nanostructure, was developed. Finite-difference time-domain numerical simulations compared favorably with experimental scatter patterns from micrometer-sized beads and cells. The device was capable of obtaining light scattering patterns from the smallest mature blood cells (platelets) and cord blood hematopoietic stem/progenitor cells 
(CD34 + cells) and myeloid precursor cells.

The ins and outs of hematopoietic stem cells: studies to improve transplantation outcomes.

Deciphering the mechanisms of hematopoietic stem/progenitor cell (HSPC) mobilization and homing is important for the development of strategies to enhance the efficacy of HSPC transplantation and achieve the full potential of HSPC-based cellular therapy. Investigation of these mechanisms has revealed interdependence among the various molecules, pathways and cellular components involved, and underscored the complex nature of these two processes. This review summarizes recent progress in identifying the specific factors implicated in HSPC mobilization and homing, with emphasis on our own work.

Innate immunity as orchestrator of stem cell mobilization.

Hematopoietic stem and progenitor cells (HSPCs), as well as other types of stem cells, circulate under steady-state conditions at detectable levels in peripheral blood (PB), with their numbers increasing in response to stress, inflammation and tissue/organ injury. This mobilization process may be envisioned as a danger-sensing response mechanism triggered by hypoxia or mechanical or infection-induced tissue damage that recruits into PB different types of stem cells that have a role in immune surveillance and organ/tissue regeneration. Mobilization is also significantly enhanced by the administration of pharmacological agents, which has been exploited in hematological transplantology as a means to obtain HSPCs for hematopoietic reconstitution.