Mesenchymal and induced pluripotent stem cell-based therapeutics: a comparison.

Stem cell-based cell therapeutics and especially those based on human mesenchymal stem cells (hMSCs) and induced pluripotent stem cells (hiPSCs) are said to have enormous developmental potential in the coming years. Their applications range from the treatment of orthopedic disorders and cardiovascular diseases to autoimmune diseases and even cancer. However, while more than 27 hMSC-derived therapeutics are currently commercially available, hiPSC-based therapeutics have yet to complete the regulatory approval process. Based on a review of the current commercially available hMSC-derived therapeutic products and upcoming hiPSC-derived products in phase 2 and 3, this paper compares the cell therapy manufacturing process between these two cell types. Moreover, the similarities as well as differences are highlighted and the resulting impact on the production process discussed. Here, emphasis is placed on (i) hMSC and hiPSC characteristics, safety, and ethical aspects, (ii) their morphology and process requirements, as well as (iii) their 2- and 3-dimensional cultivations in dependence of the applied culture medium and process mode. In doing so, also downstream processing aspects are covered and the role of single-use technology is discussed. KEY POINTS: • Mesenchymal and induced pluripotent stem cells exhibit distinct behaviors during cultivation • Single-use stirred bioreactor systems are preferred for the cultivation of both cell types • Future research should adapt and modify downstream processes to available single-use devices.

Manufacturing human mesenchymal stem cells at clinical scale: process and regulatory challenges.

Human mesenchymal stem cell (hMSC)-based therapies are of increasing interest in the field of regenerative medicine. As economic considerations have shown, allogeneic therapy seems to be the most cost-effective method. Standardized procedures based on instrumented single-use bioreactors have been shown to provide billion of cells with consistent product quality and to be superior to traditional expansions in planar cultivation systems. Furthermore, under consideration of the complex nature and requirements of allogeneic hMSC-therapeutics, a new equipment for downstream processing (DSP) was successfully evaluated. This mini-review summarizes both the current state of the hMSC production process and the challenges which have to be taken into account when efficiently producing hMSCs for the clinical scale. Special emphasis is placed on the upstream processing (USP) and DSP operations which cover expansion, harvesting, detachment, separation, washing and concentration steps, and the regulatory demands.

Cellular Agriculture: Opportunities and Challenges.

Cellular agriculture is the controlled and sustainable manufacture of agricultural products with cells and tissues without plant or animal involvement. Today, microorganisms cultivated in bioreactors already produce egg and milk proteins, sweeteners, and flavors for human nutrition as well as leather and fibers for shoes, bags, and textiles. Furthermore, plant cell and tissue cultures provide ingredients that stimulate the immune system and improve skin texture, with another precommercial cellular agriculture product, in vitro meat, currently receiving a great deal of attention. All these approaches could assist traditional agriculture in continuing to provide for the dietary requirements of a growing world population while freeing up important resources such as arable land. Despite early successes, challenges remain and are discussed in this review, with a focus on production processes involving plant and animal cell and tissue cultures.

Acetoacetate is a more efficient energy-yielding substrate for human mesenchymal stem cells than glucose and generates fewer reactive oxygen species.

Stem cells have been assumed to demonstrate a reliance on anaerobic energy generation, suited to their hypoxic in vivo environment. However, we found that human mesenchymal stem cells (hMSCs) have an active oxidative metabolism with a range of substrates. More ATP was consistently produced from substrate oxidation than glycolysis by cultured hMSCs. Strong substrate preferences were shown with the ketone body, acetoacetate, being oxidised at up to 35 times the rate of glucose. ROS-generation was 45-fold lower during acetoacetate oxidation compared with glucose and substrate preference may be an adaptation to reduce oxidative stress. The UCP2 inhibitor, genipin, increased ROS production with either acetoacetate or glucose by 2-fold, indicating a role for UCP2 in suppressing ROS production. Addition of pyruvate stimulated acetoacetate oxidation and this combination increased ATP production 27-fold, compared with glucose alone, which has implications for growth medium composition. Oxygen tension during culture affected metabolism by hMSCs. Between passages 2 and 5, rates of both glycolysis and substrate-oxidation increased at least 2-fold for normoxic (20% O2)- but not hypoxic (5% O2)-cultured hMSCs, despite declining growth rates and no detectable signs of differentiation. Culture of the cells with 3-hydroxybutyrate abolished the increased rates of these pathways. These findings have implications for stem cell therapy, which necessarily involves in vitro culture of cells, since low passage number normoxic cultured stem cells show metabolic adaptations without detectable changes in stem-like status.

Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmental defect.

BACKGROUND: Mesenchymal stem cells from adult bone marrow are multipotent cells capable of forming bone, cartilage, and other connective tissues. In a previous study, we demonstrated that autologous mesenchymal stem cells could repair a critical-sized bone defect in the dog. The objective of this study was to determine whether the use of allogeneic mesenchymal stem cells could heal a critical-sized bone defect in the femoral diaphysis in dogs without the use of immunosuppressive therapy. METHODS: A critical-sized segmental bone defect, 21 mm in length, was created in the mid-portion of the femoral diaphysis of twelve adult dogs that weighed between 22 and 25 kg. Each defect was treated with allogeneic mesenchymal stem cells loaded onto a hollow ceramic cylinder consisting of hydroxyapatite-tricalcium phosphate. A complete mismatch between donor stem cells and recipient dogs was identified by dog leukocyte antigen typing prior to implantation. The healing response was evaluated histologically and radiographically at four, eight, and sixteen weeks after implantation. The radiographic and histological results at sixteen weeks were compared with the historical data for the control defects, which included defects that had been treated with a cylinder loaded with autologous mesenchymal stem cells, defects treated with a cylinder without mesenchymal stem cells, and defects that had been left untreated (empty). The systemic immune response was evaluated by the analysis of recipient serum for production of antibodies against allogeneic cells. RESULTS: For defects treated with allogeneic mesenchymal stem cell implants, no adverse host response could be detected at any time-point. Histologically, no lymphocytic infiltration occurred and no antibodies against allogeneic cells were detected. Histologically, by eight weeks, a callus spanned the length of the defect, and lamellar bone filled the pores of the implant at the host bone-implant interface. Fluorescently labeled allogeneic cells were also detected. At sixteen weeks, new bone had formed throughout the implant. These results were consistent with those seen in implants loaded with autologous cells. Implants loaded with allogeneic or autologous stem cells had significantly greater amounts of bone within the available pore space than did cell-free implants at sixteen weeks (p < 0.05). CONCLUSIONS: The results of this study demonstrated that allogeneic mesenchymal stem cells loaded on hydroxyapatite-tricalcium phosphate implants enhanced the repair of a critical-sized segmental defect in the canine femur without the use of immunosuppressive therapy. No adverse immune response was detected in this model.

Therapeutic human cells: manufacture for cell therapy/regenerative medicine.

: Human primary cells (e.g. adult stem cells) as well as differentiated cells, including those of the immune system, have been found to be therapeutically useful and free of ethical concerns. Several products have received market authorization and numerous promising clinical trials are underway. We believe that such primary therapeutic cells will dominate the market for cell therapy applications for the foreseeable future. Consequently, production of such cellular products warrants attention and needs to be a fully controlled pharmaceutical process. Thus, where possible, such production should change from manufacture towards a truly scalable industrialized process for both allogeneic and autologous products. Here, we discuss manufacturing aspects of both autogeneic and allogeneic products, review the field, and provide historical context.

Investigation of allogeneic mesenchymal stem cell-based alveolar bone formation: preliminary findings.

This study was designed to evaluate mesenchymal stem cell (MSC)-based alveolar bone regeneration in a canine alveolar saddle defect model. MSCs were loaded onto hydroxyapatite/tricalcium phosphate (HA/TCP) matrices. Scanning electron microscopic (SEM) evaluation demonstrated greater than 75% MSC coverage of the HA/TCP porous surface prior to placement regardless of MSC donor. Matrices, 6 mm x 6 mm x 20 mm, with and without cells, were implanted for 4 and 9 weeks, then removed for histological evaluation of bone formation. Cell-free control matrices were compared with MSC-loaded matrices post implantation. Histomorphometrical analysis showed that equivalent amounts of new bone were formed within the pores of the matrices loaded with autologous MSCs or MSCs from an unrelated donor. Bone formation in the cell-free HA/TCP matrices was less extensive. There was no histologic evidence of an immunological response to autologous MSCs. Surprisingly, allogeneic MSC implantation also failed to provoke an immune response. Analysis of circulating antibody levels against MSCs supported the hypothesis that neither autologous nor allogeneic MSCs induced a systemic response by the host. Analysis of dye-labelled MSCs in histological sections confirmed that the MSCs persisted in the implants throughout the course of the experiment. At 9 weeks, labelled cells were present within the lacunae of newly formed bone. We conclude that autologous and allogeneic MSCs have the capacity to regenerate bone within craniofacial defects.