MSC-CM contains both neuroprotective and neurorestorative factors, and engages key molecular pathways involved in each of these processes (Table 14.1). These factors include NGF, insulin-like growth factor-1 (IGF-1), VEGF, glial cell-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF), bFGF, and SDF-1 or CXCL12, which bind to cognate receptors expressed on many cell types to promote survival and cellular responses involved in protection and repair [1, 39, 40, 44–48]. Selective inactivation of either BDNF or IGF-1 before administration of ASC-CM in a neonatal model of stroke significantly reduced potency [40]. As expected, IGF-1 in this context acts to protect neurons against inducers of apoptosis via a mechanism involving IGF-1-induced activation of Akt [49]. Activation of Akt provides a pro-survival signal after stroke which attenuates glutamate excitotoxicity and upregulates p-cAMP(adenosine 5′-monophosphate) response element-binding protein (CREB)/BDNF leading to increased cell survival, neurogenesis, and synaptogenesis [50–53]. Further protection is afforded by attenuation of excitotoxicity through stimulating Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways by multiple factors in CM, including BDNF, HGF, and VEGF [39, 40, 49, 54]. Dendrite formation and synaptogenesis in cultured rata hippocampal neurons were enhanced by soluble factors produced by umbilical- and BM-derived MSC. This activity was partially mediated by BDNF in the CM [55].

Neuritogenesis in PC12 cells was also enhanced by treatment with ASC-CM, which was due in part to NGF activation of AMP-activated kinase α (AMPK-α) in vitro [56, 57]. AMPK is the central energy sensor switch, and a key anti-inflammatory regulator of lipid metabolizing enzymes [58]. AMPK activation promotes neuritogenesis and axogenesis during metabolic stress and its loss in supporting structures like oligodendrocytes and astrocytes exacerbates the disease [56, 59, 60]. In the vasculature, AMPK-α1 is upstream to Akt and their cross talk promotes VEGF-induced angiogenesis [61].

Perhaps the earliest demonstration of MSC-CM potency in an animal disease model was by Kinnaird and Epstein [62]. The CM from BM-MSC promoted reperfusion of ischemic tissue in a mouse model of peripheral vascular disease. Treated tissues exhibited reduced muscular atrophy, enhanced vascularity, and reduced necrosis. A number of hypoxia-inducible pro-angiogenic and pro-survival factors were present in the CM and neutralization of two (VEGF and bFGF) only partially reduced the ability of the mixture to promote endothelial and smooth muscle cell migration. A related study by Rehman et al. demonstrated ASC expression of hypoxia-inducible trophic factors, including bFGF, VEGF, and HGF, and observed rapid positive effects after administration in a murine hind limb ischemia model [45]. Subsequently, it was determined that knockdown of HGF expression by siRNA severely attenuated ASC-mediated reperfusion in the same model [63]. In an attempt to increase BM-MSC survival and, thus increase the effective cell dose, the laboratory of Victor Dzau generated cells expressing the transgene for Akt [64]. The transgenic cells showed enhanced ability to promote repair of ischemic myocardium; it was determined that this effect was due to enhanced paracrine activity and that similar effects could be demonstrated by provision of the CM alone [13].

The CM from BM-MSC and ASC have been shown to protect and repair brain tissues in rodent models of focal and global ischemic stroke. A global ischemia neonatal rat hypoxia-ischemia encephalopathy (HIE) model was used to test ASC-CM in a neonatal model of hypoxia-ischemia (HI) injury. A single intravenous injection at up to 36 h post injury significantly reduced brain volume loss and promoted functional recovery at 1 week and 3 months [40]. Neutralization of either IGF-1 or BDNF before injection partially reduced this effect. The salutary effects of ASC-CM were related to protection from glutamate excitotoxicity and protection of neurons from ischemic insult. Similarly, a rat model of stroke induced by MCAO was treated with ASC-CM infused directly into the lateral ventricle [5]. Continuous infusion for 8 days beginning at 1 week after surgery reduced infarct volume and enhanced motor control at up to 15 days following MCAO surgery. This activity was attributed to enhanced microvascularity in the infarct zone as well as reduced cell death and inflammation with treatment. Short-term protection (e.g., 24 h) of a single intracerebroventricular injection of concentrated ASC-CM following temporary MCAO with reperfusion in a murine model was effective in reducing infarct volume and edema [7]. In a study that has implications for treatment of stroke victims with an autologous product, Tsai et al. evaluated CM from BM-MSC isolated from either normal healthy rats or animals that had experienced an induced stroke [30]. Both CM exhibited similar effectiveness in enhancing functional recovery when delivered intravenously. The effect was associated with increased neurogenesis and reduced inflammation within the infarct zone.

In the USA, an investigational new drug application (IND) is required for nearly all new drugs entering clinical trials. The IND comprises three sections: chemistry and manufacturing controls (CMC), clinical study design, and nonclinical studies. The nonclinical studies section mainly concerns safety and toxicity in animals using the clinically intended route of administration and a product very similar, if not identical, to that which will be used in the clinic. This section typically includes a description of efficacy studies in relevant disease models. The CMC section pertains to manufacturing processes and quality control systems for ensuring consistency and the absence of potentially deleterious agents in the final product. Each of the sections of the IND must provide reviewers with a sufficient amount of detail to determine the potential safety of any product before allowing evaluation in humans.

The regulatory route for licensure of an eventual drug based on MSC-CM will likely require a Biological License Application (BLA) as opposed to a New Drug Application (NDA), the latter which generally pertains to drugs of well-defined composition. Within the FDA there are two centers responsible for oversight and approval of new drugs. The Center for Biologics Evaluation and Research (CBER) and the Center for Drug Evaluation and Research (CDER). Jurisdictional oversight of biologics generally falls to CBER; with important exceptions for less complex entities, such as monoclonal antibodies and recombinant proteins. Therefore, the complexity of MSC-CM, whether whole or partially fractionated, likely will place it under the review of CBER.

The FDA, as well as other entities charged with overseeing aspects of biologic and cell therapies, has issued guidance documents detailing the requirements that must be fulfilled to create a product with appropriate characteristics (i.e., the CMC component of an IND) [65–72]. This section presents an overview of the guidelines for reagents and materials of acceptable quality for good manufacturing practices (GMP) production of MSC-CM to be used in the clinic.