Gary Brooke

Comparison of Human Placenta- and Bone Marrow–Derived Multipotent Mesenchymal Stem Cells

Bone marrow is the traditional source of human multipotent mesenchymal stem cells (MSCs), but placenta appears to be an alternative and more readily available source. This study comprehensively compared human placenta-derived MSC (hpMSC) and human bone marrow-derived MSC (hbmMSC) in terms of cell characteristics, optimal growth conditions and in vivo safety specifically to determine if hpMSC could represent a source of human MSC for clinical trial. MSC were isolated from human placenta (hpMSC) and human bone marrow (hbmMSC) and expanded ex vivo using good manufacturing practice-compliant reagents. hpMSC and hbmMSC showed similar proliferation characteristics in different basal culture media types, fetal calf serum (FCS) concentrations, FCS heat-inactivation experiments, flask types and media replacement responsiveness. However, hpMSC and hbmMSC differed with respect to their proliferation capabilities at different seeding densities, with hbmMSC proliferating more slowly than hpMSC in every experiment. hpMSC had greater long-term growth ability than hbmMSC. MSC from both sources exhibited similar light microscopy morphology, size, cell surface phenotype, and mesodermal differentiation ability with the exception that hpMSC consistently appeared less able to differentiate to the adipogenic lineage. A comparison of both hbmMSC and hpMSC from early and medium passage cultures using single-nucleotide polymorphism (SNP) GeneChip analysis confirmed GTG-banding data that no copy number changes had been acquired during sequential passaging. In three of three informative cases (in which the gender of the delivered baby was male), hpMSC were of maternal origin. Neither hpMSC nor hbmMSC caused any acute toxicity in normal mice when injected intravenously at the same, or higher, doses than those currently used in clinical trials of hbmMSC. This study suggests that human placenta is an acceptable alternative source for human MSC and their use is currently being evaluated in clinical trials.

Molecular Trafficking Mechanisms of Multipotent Mesenchymal Stem Cells Derived from Human Bone Marrow and Placenta

We compared potential trafficking mechanisms used by human (h) multipotent mesenchymal stem cells (MSC) derived from bone marrow (bm) or placenta (p). Both hbmMSC and hpMSC expressed a broad range of cell surface adhesion molecules including beta1-integrins (CD29) and CD44. Array data showed that both hbmMSC and hpMSC expressed mRNA for the cell adhesion molecules CD54 (ICAM-1), E-cadherin, CD166 (ALCAM), CD56 (NCAM), CD106 (VCAM-1), CD49a, b, c, e and f (integrins alpha1, 2, 3, 4 and 6), integrin alpha11, CD51 (integrin alphaV), and CD29 (integrins beta1). Functional binding of hpMSC, but not hbmMSC to VCAM-1 was demonstrated using recombinant chimeric constructs. Neither bone marrow nor placental MSC expressed ligands to endothelial selectins such as PSGL-1 or sialyl Lewis X (sLe(x)) carbohydrates and neither were able to bind functionally to chimeric constructs of the endothelial selectins CD62E (E-selectin) and CD62P (P-selectin). Furthermore, MSC expressed a restricted range of transferases necessary for expression of sLe(x), with no detectable expression of fucosyl transferases IV or VII. Placental MSC, but not hbmMSC, expressed mRNA for the chemokine receptors CCR1 and CCR3, and both hbmMSC and hpMSC expressed mRNA for CCR7, CCR8, CCR10, CCR11, CXCR4 and CXCR6. Intracellular chemokine receptor protein expression of CCR1, CCR3, CXCR3, CXCR4 and CXCR6 was detected in both hbmMSC and hpMSC. Cell surface expression of chemokine receptors was much more restricted with only CXCR6 displaying a strong signal on hbmMSC and hpMSC. Although cell surface expression of CXCR4 was not detected, MSC migrated in response to its ligand, CXCL12 (SDF-1). Thus, hbmMSC and hpMSC have an almost identical profile for cell surface adhesion and chemokine receptor molecules at the mRNA and protein levels. However, at the functional level, hpMSC likely utilise VLA-4-mediated binding in a superior manner to hbmMSC and thus may have superior engraftment properties to hbmMSC in vivo.

Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures

Chondrogenesis of mesenchymal stem cells (MSCs) is typically induced when they are condensed into a single aggregate and exposed to transforming growth factor-β (TGF-β). Hypoxia, like aggregation and TGF-β delivery, may be crucial for complete chondrogenesis. However, the pellet dimensions and associated self-induced oxygen gradients of current chondrogenic methods may limit the effectiveness of in vitro differentiation and subsequent therapeutic uses. Here we describe the use of embryoid body-forming technology to produce microscopic aggregates of human bone marrow MSCs (BM-MSCs) for chondrogenesis. The use of micropellets reduces the formation of gradients within the aggregates, resulting in a more homogeneous and controlled microenvironment. These micropellet cultures (~170 cells/micropellet) as well as conventional pellet cultures (~2 × 105 cells/pellet) were chondrogenically induced under 20% and 2% oxygen environments for 14 days. Compared to conventional pellets under both environments, micropellets differentiated under 2% O2 showed significantly increased sulfated glycosaminoglycan (sGAG) production and more homogeneous distribution of proteoglycans and collagen II. Aggrecan and collagen II gene expressions were increased in pellet cultures differentiated under 2% O2 relative to 20% O2 pellets but 2% O2 micropellets showed even greater increases in these genes, as well as increased SOX9. These results suggest a more advanced stage of chondrogenesis in the micropellets accompanied by more homogeneous differentiation. Thus, we present a new method for enhancing MSC chondrogenesis that reveals a unique relationship between oxygen tension and aggregate size. The inherent advantages of chondrogenic micropellets over a single macroscopic aggregate should allow for easy integration with a variety of cartilage engineering strategies.

Manufacturing of human placenta-derived mesenchymal stem cells for clinical trials.

Mesenchymal stem cells (MSC) are being used increasingly in clinical trials for a range of regenerative and inflammatory diseases. Bone marrow is the traditional source but is relatively inaccessible in large volume. MSC have now been derived from tissues other than bone marrow including placenta and adipose tissue. We have used placenta obtained after delivery as a source of MSC and have been unable to detect any marked differences from marrow‐derived MSC in terms of cell surface phenotype, chemokine receptor display, mesodermal differentiation capacity or immunosuppressive ability. This report described our manufacturing process for isolating and expanding placenta‐derived human MSC and their safe infusion into the first patient in a clinical trial program of human placenta‐derived MSC.

Comprehensive transcriptome and immunophenotype analysis of renal and cardiac MSC-like populations supports strong congruence with bone marrow MSC despite maintenance of distinct identities.

Cells resembling bone marrow mesenchymal stem cells (MSC) have been isolated from many organs but their functional relationships have not been thoroughly examined. Here we compared the immunophenotype, gene expression, multipotency and immunosuppressive potential of MSC-like colony-forming cells from adult murine bone marrow (bmMSC), kidney (kCFU-F) and heart (cCFU-F), cultured under uniform conditions. All populations showed classic MSC morphology and in vitro mesodermal multipotency. Of the two solid organ-specific CFU-F, only kCFU-F displayed suppression of T-cell alloreactivity in vitro, albeit to a lesser extent than bmMSC. Quantitative immunophenotyping using 81 phycoerythrin-conjugated CD antibodies demonstrated that all populations contained high percentages of cells expressing diagnostic MSC surface markers (Sca1, CD90.2, CD29, CD44), as well as others noted previously on murine MSC (CD24, CD49e, CD51, CD80, CD81, CD105). Illumina microarray expression profiling and bioinformatic analysis indicated a correlation of gene expression of 0.88-0.92 between pairwise comparisons. All populations expressed approximately 66% of genes in the pluripotency network (Plurinet), presumably reflecting their stem-like character. Furthermore, all populations expressed genes involved in immunomodulation, homing and tissue repair, suggesting these as conserved functions for MSC-like cells in solid organs. Despite this molecular congruence, strong biases in gene and protein expression and pathway activity were seen, suggesting organ-specific functions. Hence, tissue-derived MSC may also retain unique properties potentially rendering them more appropriate as cellular therapeutic agents for their organ of origin.

Closed system isolation and scalable expansion of human placental mesenchymal stem cells.

Mesenchymal stem cells (MSC) are emerging as a leading cellular therapy for a number of diseases. However, for such treatments to become available as a routine therapeutic option, efficient and cost‐effective means for industrial manufacture of MSC are required. At present, clinical grade MSC are manufactured through a process of manual cell culture in specialized cGMP facilities. This process is open, extremely labor intensive, costly, and impractical for anything more than a small number of patients. While it has been shown that MSC can be cultivated in stirred bioreactor systems using microcarriers, providing a route to process scale‐up, the degree of numerical expansion achieved has generally been limited. Furthermore, little attention has been given to the issue of primary cell isolation from complex tissues such as placenta. In this article we describe the initial development of a closed process for bulk isolation of MSC from human placenta, and subsequent cultivation on microcarriers in scalable single‐use bioreactor systems. Based on our initial data, we estimate that a single placenta may be sufficient to produce over 7,000 doses of therapeutic MSC using a large‐scale process. Biotechnol. Bioeng. 2012; 109:1817–1826.

Molecular Mechanisms Involved in Mesenchymal Stem Cell Migration to the Site of Acute Myocardial Infarction

Mesenchymal stem cells or multipotent mesenchymal stromal cells (both referred to as MSC) have been shown in some studies to have a beneficial effect on myocardial recovery after infarct. Current strategies for MSC delivery to heart involve intravenous, intraarterial, and intramuscular delivery. Different routes of MSC delivery and a lack of knowledge of the mechanisms that MSC utilise to migrate in vivo has most likely led to the marked variations in results that have been found. This review aims to summarise the current knowledge of MSC migratory mechanisms and looks to future methods of MSC manipulation prior to delivery in order to enhance MSC migration and engraftment.

A Defined Medium and Substrate for Expansion of Human Mesenchymal Stromal Cell Progenitors That Enriches for Osteo- and Chondrogenic Precursors

Human mesenchymal stromal cells (hMSCs) have generated significant interest due to their potential use in clinical applications. hMSCs are present at low frequency in vivo, but after isolation can be expanded considerably, generating clinically useful numbers of cells. In this study, we demonstrate the use of a defined embryonic stem cell expansion medium, mTeSR (Stem Cell Technologies), for the expansion of bone-marrow-derived hMSCs. The hMSCs grow at comparable rates, demonstrate tri-lineage differentiation potential, and show similar surface marker profiles (CD29(+), CD44(+), CD49a(+), CD73(+), CD90(+), CD105(+), CD146(+), CD166(+), CD34(-), and CD45(-)) in both the fetal bovine serum (FBS)-supplemented medium and mTeSR. However, expression of early differentiation transcription factors runt-related transcription factor 2, sex-determining region Y box 9, and peroxisome proliferator-activated receptor gamma changed significantly. Both runt-related transcription factor 2 and sex-determining region Y box 9 were upregulated, whereas peroxisome proliferator-activated receptor gamma was downregulated in mTeSR compared with FBS. Although osteogenic and chondrogenic differentiation was comparable in cells grown in mTeSR compared to FBS, adipogenic differentiation was significantly decreased in mTeSR-expanded cells, both in terms of gene expression and absolute numbers of adipocytes. The removal of the FBS from the medium and the provision of a defined medium with disclosed composition make mTeSR a superior study platform for hMSC biology in a controlled environment. Further, this provides a key step toward generating a clinical-grade medium for expansion of hMSCs for clinical applications that rely on osteo- and chondroinduction of MSCs, such as bone repair and cartilage generation.

Points to Consider in Designing Mesenchymal Stem Cell-Based Clinical Trials

Therapeutic applications of cells are likely to increase greatly in the future. Cell and cell-based gene therapy manufacturing facilities need to be purpose-designed and accredited by their national medicinal regulatory body. Production scientists need to work in close tandem with quality assurance and ethics committees to absolutely ensure the safety of new cellular products. In this review, we consider the need for preclinical safety and efficacy data, tissue source for manufacture of clinical grade human mesenchymal stem cells, aseptic tissue processing, indemnification, and the role of the national medicinal regulatory body in appropriate clinical trial design.

Manufacture of clinical grade human placenta-derived multipotent mesenchymal stromal cells.

Clinical grade human mesenchymal stem cells (MSC) are manufactured and used in clinical trials for a range of regenerative and inflammatory diseases. Human MSC have now been derived from tissues other than bone marrow, such as placenta, as described in this laboratory protocol. It provides instructions for clinical grade MSC manufacturing according to the Code of Good Manufacturing Practice (cGMP) principles and according to policies and procedures of our internal Quality Management System (QMS), which is based on the International Organization for Standardization (ISO) standard requirements. Relevant organizational structure and QMS elements are presented.