Residual Undifferentiated Cells Research Articles

Label-Free and High-Throughput Removal of Residual Undifferentiated Cells From iPSC-Derived Spinal Cord Progenitor Cells.

The transplantation of spinal cord progenitor cells (SCPCs) derived from human-induced pluripotent stem cells (iPSCs) has beneficial effects in treating spinal cord injury (SCI). However, the presence of residual undifferentiated iPSCs among their differentiated progeny poses a high risk as these cells can develop teratomas or other types of tumors post-transplantation. Despite the need to remove these residual undifferentiated iPSCs, no specific surface markers can identify them for subsequent removal. By profiling the size of SCPCs after a 10-day differentiation process, we found that the large-sized group contains significantly more cells expressing pluripotent markers. In this study, we used a sized-based, label-free separation using an inertial microfluidic-based device to remove tumor-risk cells. The device can reduce the number of undifferentiated cells from an SCPC population with high throughput (ie, >3 million cells/minute) without affecting cell viability and functions. The sorted cells were verified with immunofluorescence staining, flow cytometry analysis, and colony culture assay. We demonstrated the capabilities of our technology to reduce the percentage of OCT4-positive cells. Our technology has great potential for the "downstream processing" of cell manufacturing workflow, ensuring better quality and safety of transplanted cells.

Estimating residual undifferentiated cells in human chemically induced pluripotent stem cell derived islets using lncRNA as biomarkers

Human pluripotent stem cells (hPSCs) can generate insulin-producing beta cells for diabetes treatment, but residual undifferentiated cells may cause tumors. We developed a highly sensitive assay to detect these cells in islet cells derived from human chemically induced pluripotent stem cells (hCiPSCs), which are transgene-free and safer. We used RNA-seq data to find protein-coding and non-coding RNAs that were only expressed in hCiPSCs, not in islet cells. We confirmed these biomarkers by RT-qPCR and ddPCR. We chose long non-coding RNA (lncRNA) markers, which performed better than protein-coding RNA markers. We found that LNCPRESS2, LINC00678 and LOC105370482 could detect 1, 1 and 3 hCiPSCs in 106 islet cells by ddPCR, respectively. We tested our method on several hCiPSC lines, which could quantify 0.0001% undifferentiated cell in 106 islet cells by targeting hCiPSCs-specific lncRNA transcripts, ensuring the safety and quality of hCiPSC-derived islet cells for clinical use.

International evaluation study of a highly efficient culture assay for detection of residual human pluripotent stem cells in cell therapies.

Aim& methods: The Health and Environmental Sciences Institute Cell Therapy-TRAcking, Circulation & Safety Technical Committee launched an international, multisite study to evaluate the sensitivity and reproducibility of the highly efficient culture (HEC) assay, an in vitro assay to detect residual undifferentiated human pluripotent stem cells (hPSCs) in cell therapy products. Results: All facilities detected colonies of human induced pluripotent stem cells (hiPSCs) when five hiPSCs were spiked into 1 million hiPSC-derived cardiomyocytes. Spiking with a trace amount of hiPSCs revealed that repeatability accounts for the majority of reproducibility while the true positive rate was high. Conclusion: The results indicate that the HEC assay is highly sensitive and robust and can be generally applicable for tumorigenicity evaluation of hPSC-derived cell therapy products.

In vitro methods to ensure absence of residual undifferentiated human induced pluripotent stem cells intermingled in induced nephron progenitor cells.

Cell therapies using human induced pluripotent stem cell (hiPSC)-derived nephron progenitor cells (NPCs) are expected to ameliorate acute kidney injury (AKI). However, using hiPSC-derived NPCs clinically is a challenge because hiPSCs themselves are tumorigenic. LIN28A, ESRG, CNMD and SFRP2 transcripts have been used as a marker of residual hiPSCs for a variety of cell types undergoing clinical trials. In this study, by reanalyzing public databases, we found a baseline expression of LIN28A, ESRG, CNMD and SFRP2 in hiPSC-derived NPCs and several other cell types, suggesting LIN28A, ESRG, CNMD and SFRP2 are not always reliable markers for iPSC detection. As an alternative, we discovered a lncRNA marker gene, MIR302CHG, among many known and unknown iPSC markers, as highly differentially expressed between hiPSCs and NPCs, by RNA sequencing and quantitative RT-PCR (qRT-PCR) analyses. Using MIR302CHG as an hiPSC marker, we constructed two assay methods, a combination of magnetic bead-based enrichment and qRT-PCR and digital droplet PCR alone, to detect a small number of residual hiPSCs in NPC populations. The use of these in vitro assays could contribute to patient safety in treatments using hiPSC-derived cells.

A Non‐Destructive Electrical Assay of Stem Cell Differentiation Based on Semiconductor Biosensing

AbstractCell‐replacement therapies using human induced pluripotent stem cells (hiPSCs) with self‐renewal and pluripotency characteristics have attracted much attention in regenerative medicine. However, since residual undifferentiated cells can form tumors in transplant recipients, the deterioration of their safety after implantation needs to be resolved. In the present investigation, we developed a non‐destructive method to monitor stem cell differentiation by electrically detecting the glycoprotein biomarker podocalyxin, which is secreted from hiPSCs into cell culture supernatants using field‐effect transistor (FET) biosensors. By functionalizing the FET surface with a lectin with the small molecular size, rBC2LCN, which effectively uses a charge‐detectable region for FET‐based detection based on Debye length, the intrinsic charges of podocalyxin were successfully detected without the need for any labeling. Furthermore, it was demonstrated that the FETs monitored the decrease in the podocalyxin concentration in culture supernatants associated with the differentiation process. Our findings indicate that the FET‐based assay is a promising technique for the simple and rapid monitoring of the differentiation status of hiPSCs used for transplantation.

Treating iPSC-Derived β Cells with an Anti-CD30 Antibody-Drug Conjugate Eliminates the Risk of Teratoma Development upon Transplantation.

Insulin-producing cells derived from induced pluripotent stem cells (iPSCs) are promising candidates for β cell replacement in type 1 diabetes. However, the risk of teratoma formation due to residual undifferentiated iPSCs contaminating the differentiated cells is still a critical concern for clinical application. Here, we hypothesized that pretreatment of iPSC-derived insulin-producing cells with an anti-CD30 antibody–drug conjugate could prevent in vivo teratoma formation by selectively killing residual undifferentiated cells. CD30 is expressed in all human iPSCs clones tested by flow cytometry (n = 7) but not in iPSC-derived β cells (iβs). Concordantly, anti-CD30 treatment in vitro for 24 h induced a dose-dependent cell death (up to 90%) in human iPSCs while it did not kill iβs nor had an impact on iβ identity and function, including capacity to secrete insulin in response to stimuli. In a model of teratoma assay associated with iβ transplantation, the pretreatment of cells with anti-CD30 for 24 h before the implantation into NOD-SCID mice completely eliminated teratoma development (0/10 vs. 8/8, p < 0.01). These findings suggest that short-term in vitro treatment with clinical-grade anti-CD30, targeting residual undifferentiated cells, eliminates the tumorigenicity of iPSC-derived β cells, potentially providing enhanced safety for iPSC-based β cell replacement therapy in clinical scenarios.

Highly sensitive and non-disruptive detection of residual undifferentiated cells by measuring miRNAs in culture supernatant

The clinical usage of induced pluripotent stem cell (iPSC)-derived regenerative medicine products is limited by the possibility of residual undifferentiated cells forming tumours after transplantation. Most of the existing quality control tests involve crushing of cells. As a result, the cells to be transplanted cannot be directly tested, thereby increasing the cost of transplantation. Therefore, we tested a highly sensitive and non-disruptive quality-testing method that involves measuring microRNAs (miRNAs) in culture supernatants released by cells. By measuring miR-302b in the culture supernatant, residual iPSCs were detected with higher sensitivity than by measuring LIN28 (Lin-28 Homolog A) in the cells. To use this method, we also monitored the progression of differentiation. Our novel highly sensitive and non-disruptive method for detecting residual undifferentiated cells will contribute to reducing the manufacturing cost of iPSC-derived products and improving the safety of transplantation.

Development of an induced pluripotent stem cell–specific microRNA assay for detection of residual undifferentiated cells in natural killer cell therapy products

Most clinically evaluated chimeric antigen receptor (CAR)-based cell therapies are generated from autologous immune cells. However, there are several limitations to autologous cell therapy, including low availability, poor quality of starting cellular material and limited expansion capability. Recently, induced pluripotent stem cell (iPSC)-derived allogeneic cell therapy platforms have gained popularity, as they seek to overcome many of the challenges inherent to current autologous cell therapies. However, teratoma risk associated with residual undifferentiated cells (i.e., iPSCs) in the drug product may restrict potential clinical applications if left unaddressed. To ensure the safety of the final cell therapy product, there is a need to develop quality control assays to detect residual iPSCs. Combining microRNA (miRNA) sequencing data with publicly archived miRNA microarray datasets, we demonstrated that miRNAs belonging to the 300 family (miR-302a-5p, miR-302c-3p and miR-302d-5p) and 500 family (miR-518f-5p and miR-519-3p) were highly expressed in iPSCs (both periperal blood mononuclear cell– and T cell–derived iPSCs) compared with a number of differentiated cell types. We developed and validated a sensitive digital droplet polymerase chain reaction (ddPCR) assay targeting these miRNAs to detect low levels of residual iPSCs in differentiated cell samples. The miRNA ddPCR-based method with primers for miR-302a-5p, miR-302c-3p and miR-302d-5p detected as few as 5, 3 and 10 undifferentiated iPSCs, respectively, in the background of 106 iPSC-derived natural killer (iNK) cells. These results suggest that our method targeting identified iPSC-specific miRNA transcripts is specific and sensitive for the quality assessment of NK cell therapy products derived from iPSCs.

A selective cytotoxic adenovirus vector for concentration of pluripotent stem cells in human pluripotent stem cell-derived neural progenitor cells

Highly sensitive detection of residual undifferentiated pluripotent stem cells is essential for the quality and safety of cell-processed therapeutic products derived from human induced pluripotent stem cells (hiPSCs). We previously reported the generation of an adenovirus (Ad) vector and adeno-associated virus vectors that possess a suicide gene, inducible Caspase 9 (iCasp9), which makes it possible to sensitively detect undifferentiated hiPSCs in cultures of hiPSC-derived cardiomyocytes. In this study, we investigated whether these vectors also allow for detection of undifferentiated hiPSCs in preparations of hiPSC-derived neural progenitor cells (hiPSC-NPCs), which have been expected to treat neurological disorders. To detect undifferentiated hiPSCs, the expression of pluripotent stem cell markers was determined by immunostaining and flow cytometry. Using immortalized NPCs as a model, the Ad vector was identified to be the most efficient among the vectors tested in detecting undifferentiated hiPSCs. Moreover, we found that the Ad vector killed most hiPSC-NPCs in an iCasp9-dependent manner, enabling flow cytometry to detect undifferentiated hiPSCs intermingled at a lower concentration (0.002%) than reported previously (0.1%). These data indicate that the Ad vector selectively eliminates hiPSC-NPCs, thus allowing for sensitive detection of hiPSCs. This cytotoxic viral vector could contribute to ensuring the quality and safety of hiPSCs-NPCs for therapeutic use.

Method for selective ablation of undifferentiated human pluripotent stem cell populations for cell-based therapies.

Human pluripotent stem cells (PSCs), which are composed of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an opportunity to advance cardiac cell therapy–based clinical trials. However, an important hurdle that must be overcome is the risk of teratoma formation after cell transplantation due to the proliferative capacity of residual undifferentiated PSCs in differentiation batches. To tackle this problem, we propose the use of a minimal noncardiotoxic doxorubicin dose as a purifying agent to selectively target rapidly proliferating stem cells for cell death, which will provide a purer population of terminally differentiated cardiomyocytes before cell transplantation. In this study, we determined an appropriate in vitro doxorubicin dose that (a) eliminates residual undifferentiated stem cells before cell injection to prevent teratoma formation after cell transplantation and (b) does not cause cardiotoxicity in ESC-derived cardiomyocytes (CMs) as demonstrated through contractility analysis, electrophysiology, topoisomerase activity assay, and quantification of reactive oxygen species generation. This study establishes a potentially novel method for tumorigenic-free cell therapy studies aimed at clinical applications of cardiac cell transplantation.

Salicylic diamines selectively eliminate residual undifferentiated cells from pluripotent stem cell-derived cardiomyocyte preparations

Clinical translation of pluripotent stem cell (PSC) derivatives is hindered by the tumorigenic risk from residual undifferentiated cells. Here, we identified salicylic diamines as potent agents exhibiting toxicity to murine and human PSCs but not to cardiomyocytes (CMs) derived from them. Half maximal inhibitory concentrations (IC50) of small molecules SM2 and SM6 were, respectively, 9- and 18-fold higher for human than murine PSCs, while the IC50 of SM8 was comparable for both PSC groups. Treatment of murine embryoid bodies in suspension differentiation cultures with the most effective small molecule SM6 significantly reduced PSC and non-PSC contamination and enriched CM populations that would otherwise be eliminated in genetic selection approaches. All tested salicylic diamines exerted their toxicity by inhibiting the oxygen consumption rate (OCR) in PSCs. No or only minimal and reversible effects on OCR, sarcomeric integrity, DNA stability, apoptosis rate, ROS levels or beating frequency were observed in PSC-CMs, although effects on human PSC-CMs seemed to be more deleterious at higher SM-concentrations. Teratoma formation from SM6-treated murine PSC-CMs was abolished or delayed compared to untreated cells. We conclude that salicylic diamines represent promising compounds for PSC removal and enrichment of CMs without the need for other selection strategies.

Robust detection of undifferentiated iPSC among differentiated cells

Recent progress in human induced pluripotent stem cells (iPSC) technologies suggest that iPSC application in regenerative medicine is a closer reality. Numerous challenges prevent iPSC application in the development of numerous tissues and for the treatment of various diseases. A key concern in therapeutic applications is the safety of the cell products to be transplanted into patients. Here, we present novel method for detecting residual undifferentiated iPSCs amongst directed differentiated cells of all three germ lineages. Marker genes, which are expressed specifically and highly in undifferentiated iPSC, were selected from single cell RNA sequence data to perform robust and sensitive detection of residual undifferentiated cells in differentiated cell products. ESRG (Embryonic Stem Cell Related), CNMD (Chondromodulin), and SFRP2 (Secreted Frizzled Related Protein 2) were well-correlated with the actual amounts of residual undifferentiated cells and could be used to detect residual cells in a highly sensitive manner using qPCR. In addition, such markers could be used to detect residual undifferentiated cells from various differentiated cells, including hepatic cells and pancreatic cells for the endodermal lineage, endothelial cells and mesenchymal cells for the mesodermal lineage, and neural cells for the ectodermal lineage. Our method facilitates robust validation and could enhance the safety of the cell products through the exclusion of undifferentiated iPSC.

A technique for removing tumourigenic pluripotent stem cells using rBC2LCN lectin.

IntroductionTumourigenesis attributed to residual undifferentiated cells in a graft is considered to be a significant issue in cell therapy using human pluripotent stem cells. To ensure the safety of regenerative medicine derived from pluripotent stem cells, residual undifferentiated cells must be eliminated in the manufacturing process. We previously described the lectin probe rBC2LCN, which binds harmlessly and specifically to the cell surface of human pluripotent stem cells. We report here a technique using rBC2LCN to remove pluripotent cells from a heterogenous population to reduce the chance of teratoma formation.MethodsWe demonstrate a method for separating residual tumourigenic cells using rBC2LCN-bound magnetic beads. This technology is a novel use of their previous discovery that rBC2LCN is a lectin that selectively binds to pluripotent cells. We optimize and validate a method to remove hPSCs from a mixture with human fibroblasts using rBC2LCN-conjugated magnetic beads.ResultsCells with the potential to form teratoma could be effectively eliminated from a heterogeneous cell population with biotin-labelled rBC2LCN and streptavidin-bound magnetic beads. The efficiency was measured by FACS, ddPCR, and animal transplantation, suggesting that magnetic cell separation using rBC2LCN is quite efficient for eliminating hPSCs from mixed cell populations.ConclusionsThe removal of residual tumourigenic cells based on rBC2LCN could be a practical option for laboratory use and industrialisation of regenerative medicine using human pluripotent stem cells.

In vivo surveillance and elimination of teratoma-forming human embryonic stem cells with monoclonal antibody 2448 targeting annexin A2.

This study describes the use of a previously reported chimerised monoclonal antibody (mAb), ch2448, to kill human embryonic stem cells (hESCs) in vivo and prevent or delay the formation of teratomas. ch2448 was raised against hESCs and was previously shown to effectively kill ovarian and breast cancer cells in vitro and in vivo. The antigen target was subsequently found to be Annexin A2, an oncofetal antigen expressed on both embryonic cells and cancer cells. Against cancer cells, ch2448 binds and kills via antibody‐dependent cell‐mediated cytotoxicity (ADCC) and/or antibody‐drug conjugate (ADC) routes. Here, we investigate if the use of ch2448 can be extended to hESC. ch2448 was found to bind specifically to undifferentiated hESC but not differentiated progenitors. Similar to previous study using cancer cells, ch2448 kills hESC in vivo either indirectly by eliciting ADCC or directly as an ADC. The treatment with ch2448 post‐transplantation eliminated the in vivo circulating undifferentiated cells and prevented or delayed the formation of teratomas. This surveillance role of ch2448 adds an additional layer of safeguard to enhance the safety and efficacious use of pluripotent stem cell‐derived products in regenerative medicine. Thereby, translating the use of ch2448 in the treatment of cancers to a proof of concept study in hESC (or pluripotent stem cell [PSC]), we show that mAbs can also be used to eliminate teratoma forming cells in vivo during PSC‐derived cell therapies. We propose to use this strategy to complement existing methods to eliminate teratoma‐forming cells in vitro. Residual undifferentiated cells may escape in vitro removal methods and be introduced into patients together with the differentiated cells.

Method of localized removal of cells using a bolt-clamped Langevin transducer with an ultrasonic horn.

Cell isolation by eliminating undesirable cell aggregations or colonies with low activity is essential to improve cell culture efficiency. Moreover, when creating tissues from induced pluripotent stem cells, residual undifferentiated cells must be removed to prevent tumor formation in vivo. Here, we evaluated the use of ultrasonic irradiation, which can apply energy locally without contact, and proposed a method to eliminate cells in a small area of culture by ultrasonic irradiation from a Langevin transducer. We constructed a device that incorporated a bolt-clamped 19.84kHz Langevin transducer with an ultrasonic horn and determined the optimal conditions for stable elimination of cells in small areas of a 35-mm culture dish. The optimal conditions were as follows: number of cycles=400, clearance distance=1mm, volume of medium=4mL, and distance from the center of culture surface=0mm. The mean cell elimination area under these conditions was 0.097 mm2. We also evaluated the viability of neighboring cells after ultrasonic irradiation by fluorescent staining and found that most cells around the elimination area survived. These findings suggest that the proposed method has potential for localized elimination of cells without the need for contact with the cell surface.

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model.

The growing number of victims of "stem cell tourism," the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy. External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Discovery of Novel Cell Surface Markers for Purification of Embryonic Dopamine Progenitors for Transplantation in Parkinson's Disease Animal Models

Despite the progress in safety and efficacy of cell replacement therapy with pluripotent stem cells (PSCs), the presence of residual undifferentiated stem cells or proliferating neural progenitor cells with rostral identity remains a major challenge. Here we report the generation of a LIM homeobox transcription factor 1 alpha (LMX1A) knock-in GFP reporter human embryonic stem cell (hESC) line that marks the early dopaminergic progenitors during neural differentiation to find reliable membrane protein markers for isolation of midbrain dopaminergic neurons. Purified GFP positive cells in vitro exhibited expression of mRNA and proteins that characterized and matched the midbrain dopaminergic identity. Further quantitative proteomics analysis of enriched LMX1A+ cells identified several membrane-associated proteins including a polysialylated embryonic form of neural cell adhesion molecule (PSA-NCAM) and contactin 2 (CNTN2), enabling prospective isolation of LMX1A+ progenitor cells. Transplantation of human-PSC-derived purified CNTN2+ progenitors enhanced dopamine release from transplanted cells in the host brain and alleviated Parkinson's disease-related phenotypes in animal models. This study establishes an efficient approach for purification of large numbers of human-PSC-derived dopaminergic progenitors for therapeutic applications.

Selective Elimination of Human Induced Pluripotent Stem Cells Using Medium with High Concentration of L-Alanine

Human pluripotent stem cells, including human induced pluripotent stem cells (hiPSCs), serve as highly valuable sources for both cell-based therapies and basic research, owing to their abilities to self-renew and differentiate into any cell type of the human body. However, tumorigenic risks of residual undifferentiated stem cells limit the clinical application of hiPSCs, necessitating methods to eliminate undifferentiated hiPSCs from differentiated cells. Here, we found that undifferentiated hiPSCs were more sensitive to the treatment with a medium supplemented with high concentration of L-alanine than human fibroblasts (hFBs), human skeletal muscle cells (hSkMCs), hiPSC-derived cardiomyocytes (iCMs) or hiPSC-derived fibroblast-like cells (iFLCs), which were used as differentiated cells. Undifferentiated hiPSCs co-cultured with differentiated cells were selectively eliminated following treatment. In addition, we found that the medium supplemented with high concentration of D-alanine or β-alanine also induced cell death of hiPSCs and the treatment at 4 °C didn’t induce cell death of hiPSCs. The cell death induced would be associated partly with high osmotic pressure of the medium supplemented with L-alanine. As L-alanine is a component of proteins in human body and popular ingredient of cell culture media, treatment with high concentration of L-alanine may be useful for eliminating tumorigenic residual hiPSCs for stem cell-based therapies.

Harnessing cell pluripotency for cardiovascular regenerative medicine.

Human pluripotent stem cells (hPSCs), in particular embryonic stem cells and induced pluripotent stem cells, have received enormous attention in cardiovascular regenerative medicine owing to their ability to expand and differentiate into functional cardiomyocytes and other cardiovascular cell types. Despite the potential applications of hPSCs for tissue regeneration in patients suffering from cardiovascular disease, whether hPSC-based therapies can be safe and efficacious remains inconclusive, with strong evidence from clinical trials lacking. Critical factors limiting therapeutic efficacy are the degree of maturity and purity of the hPSC-derived differentiated progeny, and the tumorigenic risk associated with residual undifferentiated cells. In this Review, we discuss recent advances in cardiac-cell differentiation from hPSCs and in the direct reprogramming of non-myocyte cells for cardiovascular regenerative applications. We also discuss approaches for the delivery of cells to diseased tissue, and how such advances are contributing to progress in cardiac tissue engineering for tackling heart disease.

Genetic and epigenetic factors which modulate differentiation propensity in human pluripotent stem cells.

Human pluripotent stem cell (hPSC) lines are known to have a bias in their differentiation. This gives individual cell lines a propensity to preferentially differentiate towards one germ layer or cell type over others. Chromosomal aberrations, mitochondrial mutations, genetic diversity and epigenetic variance are the main drivers of this phenomenon, and can lead to a wide range of phenotypes. Our aim is to provide a comprehensive overview of the different factors which influence differentiation propensity. Specifically, we sought to highlight known genetic variances and their mechanisms, in addition to more general observations from larger abnormalities. Furthermore, we wanted to provide an up-to-date list of a growing number of predictive indicators which are able to identify differentiation propensity before the initiation of differentiation. As differentiation propensity can lead to difficulties in both research as well as clinical translation, our thorough overview could be a useful tool. Combinations of the following key words were applied as search criteria in the PubMed database: embryonic stem cells, induced pluripotent stem cells, differentiation propensity (also: potential, efficiency, capacity, bias, variability), epigenetics, chromosomal abnormalities, genetic aberrations, X chromosome inactivation, mitochondrial function, mitochondrial metabolism, genetic diversity, reprogramming, predictive marker, residual stem cell, clinic. Only studies in English were included, ranging from 2000 to 2017, with a majority ranging from 2010 to 1017. Further manuscripts were added from cross-references. Differentiation propensity is affected by a wide variety of (epi)genetic factors. These factors clearly lead to a loss of differentiation capacity, preference towards certain cell types and oftentimes, phenotypes which begin to resemble cancer. Broad changes in (epi)genetics, such as aneuploidies or wide-ranging modifications to the epigenetic landscape tend to lead to extensive, less definite changes in differentiation capacity, whereas more specific abnormalities often have precise ramifications in which certain cell types become more preferential. Furthermore, there appears to be a greater, though often less considered, contribution to differentiation propensity by factors such as mitochondria and inherent genetic diversity. Varied differentiation capacity can also lead to potential consequences in the clinical translation of hPSC, including the occurrence of residual undifferentiated stem cells, and the transplantation of potentially transformed cells. As hPSC continue to advance towards the clinic, our understanding of them progresses as well. As a result, the challenges faced become more numerous, but also more clear. If the transition to the clinic is to be achieved with a minimum number of potential setbacks, thorough evaluation of the cells will be an absolute necessity. Altered differentiation propensity represents at least one such hurdle, for which researchers and eventually clinicians will need to find solutions. Already, steps are being taken to tackle the issue, though further research will be required to evaluate any long-term risks it poses.