Therapeutic potential of ixmyelocel-T, an expanded autologous multicellular therapy for treatment of ischemic cardiovascular diseases.

INTRODUCTION: Bone marrow derived cellular therapies are an emerging approach to promoting therapeutic angiogenesis in ischemic cardiovascular disease. However, the percentage of regenerative cells in bone marrow mononuclear cells (BMMNCs) is small, and large amounts of BMMNCs are required. Ixmyelocel-T, an expanded autologous multicellular therapy, is manufactured from a small sample of bone marrow aspirate. Ixmyelocel-T contains expanded populations of mesenchymal stromal cells (MSCs) and M2-like macrophages, as well as many of the CD45+ cells found in the bone marrow. It is hypothesized that this expanded multi-cellular therapy would induce angiogenesis and endothelial repair. METHODS: A rat model of hind limb ischemia was used to determine the effects of ixmyelocel-T on blood flow recovery. To further determine the effects on endothelial cells, ixmyelocel-T was co-cultured with human umbilical vein endothelial cells (HUVEC) in non-contacting Transwell® inserts. RESULTS: Co-culture of HUVECs with ixmyelocel-T resulted secretion of a variety of pro-angiogenic factors. HUVECs stimulated by ixmyelocel-T exhibited enhanced migration, proliferation, and branch formation. Ixmyelocel-T co-culture also resulted in increased endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production. In tumor necrosis factor alpha (TNFα)-stimulated HUVECs, ixmyelocel-T co-culture decreased apoptosis and reactive oxygen species generation, increased super oxide dismutase activity, and decreased nuclear factor kappa B (NFκB) activation. Treatment with ixmyelocel-T in a rat model of hind limb ischemia resulted in significantly increased blood flow perfusion and capillary density, gene expression and plasma levels of the anti-inflammatory cytokine interleukin (IL)-10, plasma nitrates, plasma platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF) expression, and significantly decreased plasma thiobarbituric acid reactive substances (TBARS). CONCLUSIONS: This work demonstrates that ixmyelocel-T interacts with endothelial cells in a paracrine manner, resulting in angiogenesis and endothelial protection. This data suggests that ixmyelocel-T could be useful for promoting of angiogenesis and tissue repair in ischemic cardiovascular diseases. In conclusion, ixmyelocel-T therapy may provide a new aspect of therapeutic angiogenesis in this patient population where expanded populations of regenerative cells might be required.

From bench to bedside: review of gene and cell-based therapies and the slow advancement into phase 3 clinical trials, with a focus on Aastrom's Ixmyelocel-T.

There is a large body of preclinical research demonstrating the efficacy of gene and cellular therapy for the potential treatment of severe (limb-threatening) peripheral arterial disease (PAD), including evidence for growth and transcription factors, monocytes, and mesenchymal stem cells. While preclinical research has advanced into early phase clinical trials in patients, few late-phase clinical trials have been conducted. The reasons for the slow progression of these therapies from bench to bedside are as complicated as the fields of gene and cellular therapies. The variety of tissue sources of stem cells (embryonic, adult bone marrow, umbilical cord, placenta, adipose tissue, etc.); autologous versus allogeneic donation; types of cells (hematopoietic, mesenchymal stromal, progenitor, and mixed populations); confusion and stigmatism by the public and patients regarding gene, protein, and stem cell therapy; scaling of manufacturing; and the changing regulatory environment all contribute to the small number of late phase (Phase 3) clinical trials and the lack of Food and Drug Administration (FDA) approvals. This review article provides an overview of the progression of research from gene therapy to the cellular therapy field as it applies to peripheral arterial disease, as well as the position of Aastrom's cellular therapy, ixmyelocel-T, within this field.

Ixmyelocel-T, an expanded multicellular therapy, contains a unique population of M2-like macrophages.

INTRODUCTION: M2 macrophages promote tissue repair and regeneration through various mechanisms including immunomodulation and scavenging of tissue debris. Delivering increased numbers of these cells to ischemic tissues may limit tissue injury and promote repair. Ixmyelocel-T is an expanded, autologous multicellular therapy cultured from bone-marrow mononuclear cells (BMMNCs). The purpose of this study was to characterize further a unique expanded population of M2-like macrophages, generated in ixmyelocel-T therapy. METHODS: Approximately 50 ml of whole bone marrow was obtained from healthy donors and shipped overnight. BMMNCs were produced by using density-gradient separation and cultured for approximately 12 days to generate ixmyelocel-T. CD14+ cells were isolated from ixmyelocel-T with positive selection for analysis. Cell-surface phenotype was examined with flow cytometry and immunofluorescence, and expression of cytokines and chemokines was analyzed with enzyme-linked immunosorbent assay (ELISA). Quantitative real-time PCR was used to analyze expression of genes in BMMNCs, ixmyelocel-T, the CD14+ population from ixmyelocel-T, and M1 and M2 macrophages. Ixmyelocel-T was cultured with apoptotic BMMNCs, and then visualized under fluorescence microscopy to assess efferocytosis. RESULTS: Macrophages in ixmyelocel-T therapy expressed surface markers of M2 macrophages, CD206, and CD163. These cells were also found to express several M2 markers, and few to no M1 markers. After stimulation with lipopolysaccharide (LPS), they showed minimal secretion of the proinflammatory cytokines interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) compared with M1 and M2 macrophages. Ixmyelocel-T macrophages efficiently ingested apoptotic BMMNCs. CONCLUSIONS: Ixmyelocel-T therapy contains a unique population of M2-like macrophages that are characterized by expression of M2 markers, decreased secretion of proinflammatory cytokines after inflammatory stimuli, and efficient removal of apoptotic cells. This subpopulation of cells may have a potential role in tissue repair and regeneration.

Potential beneficial effects of ixmyelocel-T in the treatment of atherosclerotic diseases.

INTRODUCTION: Advanced atherosclerotic lesions are characterized by lipid accumulation, inflammation, and defective efferocytosis. An ideal therapy should address all aspects of this multifactorial disease. Ixmyelocel-T therapy, an expanded autologous multicellular therapy showing clinical promise in the treatment of diseases associated with advanced atherosclerosis, includes a novel population of M2-like macrophages. Here, we examine the macrophages of ixmyelocel-T and determine their ability to influx modified cholesterol in an atheroprotective manner, maintaining cholesterol homeostasis and preventing cellular dysfunction and death, ultimately promoting reverse cholesterol efflux. METHODS: Approximately 50 ml of whole bone marrow was obtained from healthy donors and shipped overnight. Bone marrow mononuclear cells (BMMNCs) were produced by using density gradient separation and cultured for approximately 12 days to generate ixmyelocel-T. CD14+ cells were isolated from ixmyelocel-T via positive selection for analysis. Ixmyelocel-T and human leukemia monocyte (THP-1) cells were loaded with acetylated low-density lipoprotein (Ac-LDL) for analysis. Flow cytometry and immunofluorescence were used to examine Ac-LDL uptake, expression of cytokines was analyzed by enzyme-linked immunofluorescence assay (ELISA), and quantitative real-time PCR was used to analyze expression of cholesterol-transport genes. Both the in vitro cholesterol efflux assay and in vivo reverse cholesterol transport assay were used to examine cholesterol transport. RESULTS: Ixmyelocel-T macrophages take up acetylated low-density lipoprotein and express the scavenger receptors CD36 and scavenger receptor-B1 (SR-B1). Ixmyelocel-T did not become apoptotic or proinflammatory after lipid loading. The cholesterol transporter genes ABAC1 and ABCG1 were both statistically significantly upregulated when ixmyelocel-T macrophages were loaded with cholesterol. Ixmyelocel-T also exhibited enhanced apolipoprotein A-I (ApoAI)-mediated cholesterol efflux. In addition, in vivo reverse cholesterol-transport assay demonstrated that ixmyelocel-T was able to efflux cholesterol in this model. CONCLUSIONS: Ixmyelocel-T macrophages influx modified cholesterol, remained anti-inflammatory in the face of lipid loading and inflammatory challenge, and displayed enhanced cholesterol efflux capabilities. These combined features suggest that this autologous multicellular therapy may exert beneficial effects in atherosclerotic diseases.

The Aastrom experience.

Aastrom Biosciences has developed a proprietary cell-processing technology that enables the manufacture of ixmyelocel-T, a patient-specific multicellular therapy expanded from a small sample of a patient's own bone marrow. Ixmyelocel-T is produced under current good manufacturing practices (cGMP) in a fully closed, automated system that expands mesenchymal stem cells (MSCs) and macrophages. While the cell types in ixmyelocel-T are the same as those found in the bone marrow, the numbers of MSCs and alternative macrophages are greater in ixmyelocel-T. We propose that the mixture of expanded MSCs and alternatively activated macrophages promote long-term tissue repair of ischemic tissue. The multiple cell types in ixmyelocel-T have a range of biological activities that are likely to contribute to a complex mechanism of action. Clinical trial data collected to date support the potential for ixmyelocel-T as an efficacious and safe treatment for ischemic cardiovascular indications, including critical limb ischemia (CLI) and a severe form of heart failure, dilated cardiomyopathy (DCM). The CLI clinical program has completed phase 2 and has reached concurrence with the Food and Drug Administration (FDA) on a phase 3 study (REVIVE) through the Special Protocol Assessment (SPA) process. The phase 3 study began screening patients in February 2012. The DCM clinical program will initiate phase 2b in 2012.

A 3-dimensional extracellular matrix as a delivery system for the transplantation of glioma-targeting neural stem/progenitor cells.

Neural stem/progenitor cells (NSPCs) display inherent pathotropic properties that can be exploited for targeted delivery of therapeutic genes to invasive malignancies in the central nervous system. Optimizing transplantation efficiency will be essential for developing relevant NSPC-based brain tumor therapies. To date, the real-world issue of handling and affixing NSPCs in the context of the neurosurgical resection cavity has not been addressed. Stem cell transplantation using biocompatible devices is a promising approach to counteract poor NSPC graft survival and integration in various types of neurological disorders. Here, we report the development of a 3-dimensional substrate that is based on extracellular matrix purified from tissue-engineered skin cultures (3DECM). 3DECM enables the expansion of embedded NSPCs in vitro while retaining their uncommitted differentiation status. When implanted in intracerebral glioma models, NSPCs were able to migrate out of the 3DECM to targeted glioma growing in the contralateral hemisphere, and this was more efficient than the delivery of NSPC by intracerebral injection of cell suspensions. Direct application of a 3DECM implant into a tumor resection cavity led to a marked NSPC infiltration of recurrent glioma. The semisolid consistency of the 3DECM implants allowed simple handling during the surgical procedure of intracerebral and intracavitary application and ensured continuous contact with the surrounding brain parenchyma. Here, we demonstrate proof-of-concept of a matrix-supported transplantation of tumor-targeting NSPC. The semisolid 3DECM as a delivery system for NSPC has the potential to increase transplantation efficiency by reducing metabolic stress and providing mechanical support, especially when administered to the surgical resection cavity after brain tumor removal.

Isolation of oligodendroglial cells from cultured neural stem/progenitors.

The central nervous system (CNS) is composed of multiple cell types formed through a process of lineage commitment and phenotypic differentiation of stem-like progenitor cells into three key cell types: neurons, astrocytes, and oligodendrocytes. The ability to isolate and culture these CNS stem/progenitors has facilitated the characterization of the molecular mechanisms that regulate this process, in the hopes of providing therapeutically effective cells to treat disease and injury. Although astroglial, and to a lesser extent some neuronal, phenotypes are robustly generated when these cultured stem/progenitor cells are induced to differentiate, oligodendrocytes that form the myelin-rich sheath that allows nerves to conduct action potentials are only formed at a low frequency. This relatively low frequency has necessitated the development of methods for quantifying oligodendroglial phenotypes in vitro, with greater precision and accuracy than the standard technique of microscopic counting by hand. Here, we describe the isolation of neural stem cells and the application of intracellular flow cytometry to quantify oligodendroglial phenotypes in cultured CNS stem/progenitor cells using commercially available kits.