Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or $\$-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

Pluripotent stem cells can become any cell type found in the body. Accordingly, one of the major challenges when working with pluripotent stem cells is producing a highly homogenous population of differentiated cells, which can then be used for downstream applications such as cell therapies or drug screening. The transcription factor Ascl1 plays a key role in neural development and previous work has shown that Ascl1 overexpression using viral vectors can reprogram fibroblasts directly into neurons. Here we report on how a recombinant version of the Ascl1 protein functionalized with intracellular protein delivery technology (Ascl1-IPTD) can be used to rapidly differentiate human induced pluripotent stem cells (hiPSCs) into neurons. We first evaluated a range of Ascl1-IPTD concentrations to determine the most effective amount for generating neurons from hiPSCs cultured in serum free media. Next, we looked at the frequency of Ascl1-IPTD supplementation in the media on differentiation and found that one time supplementation is sufficient enough to trigger the neural differentiation process. Ascl1-IPTD was efficiently taken up by the hiPSCs and enabled rapid differentiation into TUJ1-positive and NeuN-positive populations with neuronal morphology after 8 days. After 12 days of culture, hiPSC-derived neurons produced by Ascl1-IPTD treatment exhibited greater neurite length and higher numbers of branch points compared to neurons derived using a standard neural progenitor differentiation protocol. This work validates Ascl1-IPTD as a powerful tool for engineering neural tissue from pluripotent stem cells.

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.