Predicting differentiation potential of human pluripotent stem cells: Possibilities and challenges

Abstract

The capability of human pluripotent stem cell (hPSC) lines to propagate indefinitely and differentiate into derivatives of three embryonic germ layers makes these cells be powerful tools for basic scientific research and promising agents for translational medicine. However, variations in differentiation tendency and efficiency as well as pluripotency maintenance necessitate the selection of hPSC lines for the intended applications to save time and cost. To screen the qualified cell lines and exclude problematic cell lines, their pluripotency must be confirmed initially by traditional methods such as teratoma formation or by high-throughput gene expression profiling assay. Additionally, their differentiation potential, particularly the lineage-specific differentiation propensities of hPSC lines, should be predicted in an early stage. As a complement to the teratoma assay, RNA sequencing data provide a quantitative estimate of the differentiation ability of hPSCs in vivo . Moreover, multiple scorecards have been developed based on selected gene sets for predicting the differentiation potential into three germ layers or the desired cell type many days before terminal differentiation. For clinical application of hPSCs, the malignant potential of the cells must also be evaluated. A combination of histologic examination of teratoma with quantitation of gene expression data derived from teratoma tissue provides safety-related predictive information by detecting immature teratomas, malignancy marker expression, and other parameters. Although various prediction methods are available, distinct limitations remain such as the discordance of results between different assays and requirement of a long time and high labor and cost, restricting their wide applications in routine studies. Therefore, simpler and more rapid detection assays with high specificity and sensitivity that can be used to monitor the status of hPSCs at any time and fewer targeted markers that are more specific for a given desired cell type are urgently needed.

Enhanced differentiation of human induced pluripotent stem cells toward the midbrain dopaminergic neuron lineage through GLYPICAN-4 downregulation

“…Next, we analyzed the pluripotent differentiation potential of GPC4sh hiPSCs in vitro and in vivo by performing embryoid body (EB)‐mediated spontaneous differentiation and teratoma formation assays, respectively 29 . By using immunocytochemistry, we found that differentiating GPC4sh EBs exhibit loss of the hiPSC marker OCT4 and express markers of the three germ layers, such as OTX2 for Neuroectoderm, BRA for Mesoderm and SOX17 for Endoderm, similar to controls (Figures 1K and S2I).…”

Neurothreads: Cryogel carrier-based differentiation and delivery of mature neurons in the treatment of Parkinson’s disease

“…Additionally, there is a risk of debilitating or life-threatening side-effects, such as tumorigenesis -due to remaining proliferating cells -as well as graft-induced dyskinesias (GIDs) of unknown origin, previously observed in some patients with fetal transplants 2,4 . The differentiation and malignancy potential of a given iPSC line remains difficult to predict 17 , and pretransplantation quality assessment at later time points can be expected to reveal more pertinent information regarding the in-vivo outcome. Mature neurons however have an overly complex and fragile architecture, and thus are poorly tolerant to pre-transplantational handling procedures, particularly enzymatic dissociation 4,18 .…”

Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research

“…Directed differentiation, on the contrary, might test very specific pathways, and the optimal assay to use is still under discussion (The International Stem Cell Initiative, 2018). For a predictive assay, most probably directed differentiation into the cell type of interest is the most meaningful readout (Liu et al, 2019).…”