The Aastrom experience

Abstract

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.

Figure 1

Aastrom manufacturing platform. Left: single-use cell cassette. Right: highly automated instrument platform.

Figure 2

Changes in cell types during the culture process for ixmyelocel-T. (a) Fold change in cell phenotypes of the expanded and depleted cells in ixmyelocel-T. (b) Comparison of bone marrow mononuclear cells (BMMNCs) and ixmyelocel-T from a matched donor by flow cytometry. (c) Kinetics of expansion of CD90⁺ mesenchymal stem cells and CD14⁺Auto⁺ activated macrophages and overall loss of CD45 cells over time during ixmyelocel-T. (d) Immunofluorescence image of CD90 (red), CD14 (green), and nuclei (blue) at 600×. FACS, fluorescence-activated cell sorting.

Figure 3

Ixmyelocel-T cell phenotypes. NK, natural killer.

Figure 4

Fluorescence-activated cell sorting analysis for acetylated low-density lipoprotein (AcLDL) uptake shows ixmyelocel-T macrophages are active phagocytes. The phagocytic cells that are Ac-LDL⁺ (black arrow) are all CD14⁺ (red arrow).

Figure 5

Ixmyelocel-T CD14⁺ cells co-express CD206 and CD163.

Figure 6

Summary of the multiplex cytokine analysis of bone marrow mononuclear cells (BMMNCs) compared to ixmyelocel-T CD90⁺ and CD14⁺Auto populations. Cells were cultured for approximately 24 hours in multiwell plates and cytokine levels were measured in comparison to blank medium negative controls. CD90⁺ and CD14⁺Auto⁺ populations were sorted using fluorescence-activated cell sorting to high purity prior to culture. Non-paired data from two or more independent donors assayed in replicate are expressed as the mean cytokine concentration ± standard error in pg/ml. IL-1ra, IL-1 receptor antagonist; HGF, hepatocyte growth factor; MIP, macrophage inflammatory protein; VEGF, vascular endothelial growth factor.

Figure 7

Flow diagram of the IMPACT-DCM and CATHETER-DCM studies. DCM, dilated cardiomyopathy.

Figure 8

Flow diagram of the RESTORE-CLI study.

Figure 9

Kaplan-Meier curve: time to treatment failure (RESTORE-CLI). Kaplan-Meier survival plot of time to treatment failure (major amputation of injected leg, all-cause mortality, doubling of total wound surface area from baseline, de novo gangrene) for all patients injected. Censored observations are indicated by plus symbols. CL, confidence limit; NA, not available. Reprinted with permission of the author [21].