Richard Schäfer

Intranasal Delivery of Bone Marrow-Derived Mesenchymal Stem Cells, Macrophages, and Microglia to the Brain in Mouse Models of Alzheimer's and Parkinson's Disease

In view of the rapid preclinical development of cell-based therapies for neurodegenerative disorders, traumatic brain injury, and tumors, the safe and efficient delivery and targeting of therapeutic cells to the central nervous system is critical for maintaining therapeutic efficacy and safety in the respective disease models. Our previous data demonstrated therapeutically efficacious and targeted delivery of mesenchymal stem cells (MSCs) to the brain in the rat 6-hydroxydopamine model of Parkinsons disease (PD). The present study examined delivery of bone marrow-derived MSCs, macrophages, and microglia to the brain in a transgenic model of PD [(Thy1)-h[A30P] aS] and an APP/PS1 model of Alzheimers disease (AD) via intranasal application (INA). INA of microglia in naive BL/6 mice led to targeted and effective delivery of cells to the brain. Quantitative PCR analysis of eGFP DNA showed that the brain contained the highest amount of eGFP-microglia (up to 2.1 × 104) after INA of 1 × 106 cells, while the total amount of cells detected in peripheral organs did not exceed 3.4 × 103. Seven days after INA, MSCs expressing eGFP were detected in the olfactory bulb (OB), cortex, amygdala, striatum, hippocampus, cerebellum, and brainstem of (Thy1)-h[A30P] aS transgenic mice, showing predominant distribution within the OB and brainstem. INA of eGFP-expressing macrophages in 13-month-old APP/PS1 mice led to delivery of cells to the OB, hippocampus, cortex, and cerebellum. Both MSCs and macrophages contained Iba-1-positive population of small microglia-like cells and Iba-1-negative large rounded cells showing either intracellular amyloid β (macrophages in APP/PS1 model) or α-synuclein [MSCs in (Thy1)-h[A30P] aS model] immunoreactivity. Here, we show, for the first time, intranasal delivery of cells to the brain of transgenic PD and AD mouse models. Additional work is needed to determine the optimal dosage (single treatment regimen or repeated administrations) to achieve functional improvement in these mouse models with intranasal microglia/macrophages and MSCs. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

Unraveling the Mesenchymal Stromal Cells' Paracrine Immunomodulatory Effects

In the last 10 years, the role of mesenchymal stromal cells (MSCs) in modulating inflammatory and immune responses has been characterized using both in vitro studies and in vivo models of immune disorders. Mesenchymal stromal cell immunomodulatory properties have been linked to various paracrine factors which expression varies depending on the pathologic condition to which the MSCs are exposed. These factors may directly impact key cells of the adaptive immune system, such as T cells. Indeed, coculturing MSCs with T cells in a mixed lymphocyte reaction assay inhibits T-cell proliferation through the secretion of immunomodulatory cytokines. However, in a context of inflammation, MSCs may secrete paracrine factors that influence other immune cell subpopulations such as dendritic cells and macrophages and polarize them toward a tolerogenic phenotype. In vivo, these same immunomodulatory factors are shown to be increased in the serum of animal models presenting with inflammatory diseases treated with MSC administration. In light of the results from these landmark studies, we review the main MSC secreted factors identified to play a role in modulating inflammatory immune responses either in vitro or in vivo, and we assess the impact of these factors on the therapeutic applications of MSC-based cell therapies in immune diseases.

Platelet lysate as a substitute for animal serum for the ex-vivo expansion of mesenchymal stem/stromal cells: present and future.

The use of fetal bovine serum (FBS) as a cell culture supplement is discouraged by regulatory authorities to limit the risk of zoonoses and xenogeneic immune reactions in the transplanted host. Additionally, FBS production came under scrutiny due to animal welfare concerns. Platelet derivatives have been proposed as FBS substitutes for the ex-vivo expansion of mesenchymal stem/stromal cells (MSCs) since platelet-derived growth factors can promote MSC ex-vivo expansion. Platelet-derived growth factors are present in platelet lysate (PL) obtained after repeated freezing–thawing cycles of the platelet-rich plasma or by applying physiological stimuli such as thrombin or CaCl2.PL-expanded MSCs have been used already in the clinic, taking advantage of their faster proliferation compared with FBS-expanded preparations. Should PL be applied to other biopharmaceutical products, its demand is likely to increase dramatically. The use of fresh platelet units for the production of PL raises concerns due to limited availability of platelet donors. Expired units might represent an alternative, but further data are needed to define safety, including pathogen reduction, and functionality of the obtained PL. In addition, relevant questions concerning the definition of PL release criteria, including concentration ranges of specific growth factors in PL batches for various clinical indications, also need to be addressed. We are still far from a common definition of PL and standardized PL manufacture due to our limited knowledge of the mechanisms that mediate PL-promoting cell growth. Here, we concisely discuss aspects of PL as MSC culture supplement as a preliminary step towards an agreed definition of the required characteristics of PL for the requirements of manufacturers and users.

Cell-based therapies for cardiac disease: a cellular therapist's perspective.

Cell‐based therapy is an exciting, promising, and a developing new treatment for cardiac diseases. Stem cell–based therapies have the potential to fundamentally transform the treatment of ischemic cardiac injury and heart failure by achieving what would have been unthinkable only a few years ago—the Holy Grail of myocardial regeneration. Recent therapeutic approaches involve bone marrow (BM)‐derived mononuclear cells and their subsets such as mesenchymal stem/stromal cells (MSCs), endothelial progenitor cells as well as adipose tissue–derived MSCs, cardiac tissue–derived stem cells, and cell combinations. Clinical trials employing these cells have demonstrated that cellular therapy is feasible and safe. Regarding delivery methods, the safety of catheter‐based, transendocardial and ‐epicardial stem cell injection has been established. However, the results, while variable, suggest rather modest clinical efficacy overall in both heart failure and ischemic heart disease, such as in acute myocardial infarction. Future studies will focus on determining the most efficacious cell type(s) and/or cell combinations and the most reasonable indications and optimal timing of transplantation, as well as the mechanisms underlying their therapeutic effects. We will review and summarize the clinical trial results to date. In addition, we discuss challenges and operational issues in cell processing for cardiac applications.

Where is the common ground between bone marrow mesenchymal stem/stromal cells from different donors and species?

Mesenchymal stem/stromal cells (MSCs) feature promising potential for cellular therapies, yet significant progress in development of MSC therapeutics and assays is hampered because of remarkable MSC heterogeneity in vivo and in vitro. This heterogeneity poses challenges for standardization of MSC characterization and potency assays as well as for MSC study comparability and manufacturing. This review discusses promising marker combinations for prospective MSC subpopulation enrichment and expansion, and reflects MSC phenotype changes due to environment and age. In order to address animal modelling in MSC biology, comparison of mouse and human MSC markers highlights current common ground of MSCs between species.

Basic research and clinical applications of non-hematopoietic stem cells, 4–5 April 2008, Tübingen, Germany

From 4 to 5 April 2008, international experts met for the second time in Tubingen, Germany, to present and discuss the latest proceedings in research on non-hematopoietic stem cells (NHSC). This report presents issues of basic research including characterization, isolation, good manufacturing practice (GMP)-like production and imaging as well as clinical applications focusing on the regenerative and immunomodulatory capacities of NHSC.

A Simple "Blood-Saving Bundle" Reduces Diagnostic Blood Loss and the Transfusion Rate in Mechanically Ventilated Patients.

Introduction Aim of this study was to reduce blood loss caused by diagnostic blood sampling and to minimize the development of anemia in a high-risk group of mechanically ventilated medical intensive care patients. We therefore implemented a “blood-saving bundle” (BSB) combining a closed-loop arterial blood sampling system, smaller sampling tubes, reduced frequency of blood drawings, and reduced sample numbers. Methods The study included all patients from our medical ICU who were ventilated for more than 72 hours. Exclusion criteria were: acute or chronic anemia on admission, bleeding episode(s) during the ICU stay, or end-of-life therapy. The BSB was introduced in 2009 with training and educational support. Patients treated in 2008, before the introduction of the BSB, served as a control group (n = 41, 617 observation days), and were compared with patients treated in 2010 after the introduction of the BSB (BSB group, n = 50, 559 observation days). Primary endpoints were blood loss per day, and development of anemia. Secondary endpoints were numbers of blood transfusions, number of days on mechanical ventilation, and length of the ICU stay. Results Mean blood loss per ICU day was decreased from 43.3 ml (95% CI: 41.2 to 45.3 ml) in the controls to 15.0 ml (14.3 to 15.7 ml) in the BSB group (P < 0.001). The introduction of a closed-loop arterial blood sampling system was the major contributor to this effect. Mean hemoglobin concentrations showed no significant differences in both groups during the ICU stay. Hemoglobin values <9 g/dl, however, were recorded in 21.2% of observation days in the controls versus 15.4% in the BSB group (P = 0.01). Units of transfused red blood cells per 100 observation days decreased from 7 to 2.3 (P < 0.001). The mean number of ventilation days was 7.1 days (6.1 to 8.3 days) in the controls and 7.5 days (6.6 to 8.5 days) in the BSB group (P = NS). In total, patients in the BSB group stayed in ICU for a mean of 9.9 days (8.6 to 11.3 days), compared to a mean ICU stay of 13.0 days (10.9 to 15.4 days) in the control group (P = 0.014). Due to the longitudinal study design, however, we cannot exclude uncontrolled confounders affecting the transfusion frequency and mean ICU stay. Conclusion Our BSB could be easily implemented and was able to reduce diagnostic blood loss.

Effective treatment of steroid and therapy-refractory acute graft-versus-host disease with a novel mesenchymal stromal cell product (MSC-FFM)

The inability to generate mesenchymal stromal cells (MSCs) of consistent potency likely is responsible for inconsistent clinical outcomes of patients with aGvHD receiving MSC products. We developed a novel MSC manufacturing protocol characterized by high in vitro potency and near-identity of individual doses, referred to as “MSC-Frankfurt am Main (MSC-FFM)”. Herein, we report outcomes of the 69 patients who have received MSC-FFM. These were 51 children and 18 adults with refractory aGvHD grade II (4%), III (36%) or IV (59%). Patients were refractory either to frontline therapy (steroids) (29%) or to steroids and 1–5 additional lines of immunosuppressants (71%) were given infusions in four weekly intervals. The day 28 overall response rate was 83%; at the last follow-up, 61% and 25% of patients were in complete or partial remission. The median follow-up was 8.1 months. Six-month estimate for cumulative incidence of non-relapse mortality was 27% (range, 16–38); leukemia relapse mortality was 2% (range, 0–5). This was associated with a superior six-month overall survival (OS) probability rate of 71% (range, 61–83), compared to the outcome of patients not treated with MSC-FFM. This novel product was effective in children and adults, suggesting that MSC-FFM represents a promising therapy for steroid refractory aGvHD.

Manufacture of endothelial colony-forming progenitor cells from steady-state peripheral blood leukapheresis using pooled human platelet lysate: ECFC MANUFACTURING FROM LEUKAPHERESIS

Endothelial colony‐forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP).

Characterization of mesenchymal stem or stromal cells: tissue sources, heterogeneity, and function.

I t appears that much progress has been made since Alexander Friedenstein’s first descriptions of bone marrow (BM) stromal cells, variably described in the following years as mesenchymal stem or stromal cells (MSCs) and featuring high in vitro replicative capacity as well as osteogenic and chondrogenic differentiation potential. However, despite decades of research leading to a fair understanding of role of MSCs in maintenance of the hematopoietic niche, it is the obvious heterogeneity of MSCs that has been posing an extraordinary challenge for our understanding of MSCs biology and their translation to the clinic. In general, MSC heterogeneity comes on various levels: