Simple SummaryAlzheimer’s Disease (AD) is a leading cause of death and disability worldwide. Unfortunately, medical researchers have struggled to develop methods to understand why some people develop AD while others do not while also focusing on the disease’s late-stage consequences. We wanted to address this problem by modeling the disease using human-like physiological conditions. We wanted to explore the properties of human neural stem cells (NSCs) because these cells develop and maintain brain tissue. Conventional xenogeneic cultures cannot accurately recapitulate AD in vitro, and we needed a new model that would better represent the human brain’s structure, function, and pathology to study AD more effectively. We built a novel model using human induced pluripotent stem (iPS) cells combined with an extracellular scaffold of protein derived from human products and compared its basic stem cell properties versus the standard mouse-derived scaffold. We found different stemness and differentiation properties from cell lines cultured on the scaffolds, with more human-like characteristics found when using the human-derived scaffold. Our xeno-free model more closely mimics the human brain microenvironment, potentially increasing the quality of AD research. This is a technological advancement that the field can use in research experiments to understand AD better and to more effectively develop and test drugs and therapies to prevent and manage this terrible disease.AbstractAlzheimer’s Disease (AD) is characterized by synapse and neuronal loss and the accumulation of neurofibrillary tangles and Amyloid β plaques. Despite significant research efforts to understand the late stages of the disease, its etiology remains largely unknown. This is in part because of the imprecise AD models in current use. In addition, little attention has been paid to neural stem cells (NSC), which are the cells responsible for the development and maintenance of brain tissue during an individual’s lifespan. Thus, an in vitro 3D human brain tissue model using induced pluripotent stem (iPS) cell-derived neural cells in human physiological conditions may be an excellent alternative to standard models to investigate AD pathology. Following the differentiation process mimicking development, iPS cells can be turned into NSCs and, ultimately, neural cells. During differentiation, the traditionally used xenogeneic products may alter the cells’ physiology and prevent accurate disease pathology modeling. Hence, establishing a xenogeneic material-free cell culture and differentiation protocol is essential. This study investigated the differentiation of iPS cells to neural cells using a novel extracellular matrix derived from human platelet lysates (PL Matrix). We compared the stemness properties and differentiation efficacies of iPS cells in a PL matrix against those in a conventional 3D scaffold made of an oncogenic murine-matrix. Using well-defined conditions without xenogeneic material, we successfully expanded and differentiated iPS cells into NSCs via dual-SMAD inhibition, which regulates the BMP and TGF signaling cascades in a manner closer to human conditions. This in vitro, 3D, xenogeneic-free scaffold will enhance the quality of disease modeling for neurodegenerative disease research, and the knowledge produced could be used in developing more effective translational medicine.