Sean P. Sherman

Female Human iPSCs Retain an Inactive X Chromosome

Generating induced pluripotent stem cells (iPSCs) requires massive epigenome reorganization. It is unclear whether reprogramming of female human cells reactivates the inactive X chromosome (Xi), as in mouse. Here we establish that human (h)iPSCs derived from several female fibroblasts under standard culture conditions carry an Xi. Despite the lack of reactivation, the Xi undergoes defined chromatin changes, and expansion of hiPSCs can lead to partial loss of XIST RNA. These results indicate that hiPSCs are epigenetically dynamic and do not display a pristine state of X inactivation with two active Xs as found in some female human embryonic stem cell lines. Furthermore, whereas fibroblasts are mosaic for the Xi, hiPSCs are clonal. This nonrandom pattern of X chromosome inactivation in female hiPSCs, which is maintained upon differentiation, has critical implications for clinical applications and disease modeling, and could be exploited for a unique form of gene therapy for X-linked diseases.

Involvement of Lysosomal Exocytosis in the Excretion of Mesoporous Silica Nanoparticles and Enhancement of the Drug Delivery Effect by Exocytosis Inhibition

The exocytosis of phosphonate modified mesoporous silica nanoparticles (P-MSNs) is demonstrated and lysosomal exocytosis is identified as the mechanism responsible for this event. Regulation of P-MSN exocytosis can be achieved by inhibiting or accelerating lysosomal exocytosis. Slowing down P-MSN exocytosis enhances the drug delivery effect of CPT-loaded P-MSNs by improving cell killing.

Integrated Chemical Genomics Reveals Modifiers of Survival in Human Embryonic Stem Cells

Understanding how survival is regulated in human embryonic stem cells (hESCs) could improve expansion of stem cells for production of cells for regenerative therapy. There is great variability in comparing the differentiation potential of multiple hESC lines. One reason for this is poor survival upon dissociation, which limits selection of homogeneous populations of cells. Understanding the complexity of survival signals has been hindered by the lack of a reproducible system to identify modulators of survival in pluripotent cells. We therefore developed a high‐content screening approach with small molecules to examine hESC survival. We have identified novel small molecules that improve survival by inhibiting either Rho‐kinase or protein kinase C. Importantly, small molecule targets were verified using short hairpin RNA. Rescreening with stable hESCs that were genetically altered to have increased survival enabled us to identify groups of pathway targets that are important for modifying survival. Understanding how survival is regulated in hESCs could overcome severe technical difficulties in the field, namely expansion of stem cells to improve production of cells and tissues for regenerative therapy. STEM CELLS 2009;27:533–542

Nipah Virus Envelope-Pseudotyped Lentiviruses Efficiently Target ephrinB2-Positive Stem Cell Populations In Vitro and Bypass the Liver Sink When Administered In Vivo

ABSTRACT Sophisticated retargeting systems for lentiviral vectors have been developed in recent years. Most seek to suppress the viral envelopes natural tropism while modifying the receptor-binding domain such that its tropism is determined by the specificity of the engineered ligand-binding motif. Here we took advantage of the natural tropism of Nipah virus (NiV), whose attachment envelope glycoprotein has picomolar affinity for ephrinB2, a molecule proposed as a molecular marker of “stemness” (present on embryonic, hematopoietic, and neural stem cells) as well as being implicated in tumorigenesis of specific cancers. NiV entry requires both the fusion (F) and attachment (G) glycoproteins. Truncation of the NiV-F cytoplasmic tail (T5F) alone, combined with full-length NiV-G, resulted in optimal titers of NiV-pseudotyped particles (NiVpp) (∼106 IU/ml), even without ultracentrifugation. To further enhance the infectivity of NiVpp, we engineered a hyperfusogenic NiV-F protein lacking an N-linked glycosylation site (T5FΔN3). T5FΔN3/wt G particles exhibited enhanced infectivity on less permissive cell lines and efficiently targeted ephrinB2+ cells even in a 1,000-fold excess of ephrinB2-negative cells, all without any loss of specificity, as entry was abrogated by soluble ephrinB2. NiVpp also transduced human embryonic, hematopoietic, and neural stem cell populations in an ephrinB2-dependent manner. Finally, intravenous administration of the luciferase reporter NiVpp-T5FΔN3/G to mice resulted in signals being detected in the spleen and lung but not in the liver. Bypassing the liver sink is a critical barrier for targeted gene therapy. The extraordinary specificity of NiV-G for ephrinB2 holds promise for targeting specific ephrinB2+ populations in vivo or in vitro.

A spatially and chemically defined platform for the uniform growth of human pluripotent stem cells.

We report the design of a chemically defined platform engineered for the culture of human pluripotent stem cells (hPSCs) that supports the long-term maintenance of self-renewing hPSC populations in a more uniform manner than standard culture systems. Microcontact printing (μCP) of alkanethiol self-assembled monolayers (SAMs) was used to spatially direct hPSC adherence. This technique not only establishes control over hPSC colony size and shape but also preserves genetic stability and provides unprecedented uniformity in the pluripotency of hPSC populations that is quantitatively assessed in the present study.

Small Molecule Screening with Laser Cytometry Can Be Used to Identify Pro-Survival Molecules in Human Embryonic Stem Cells

Differentiated cells from human embryonic stem cells (hESCs) provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs) provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.

High-throughput screen for compounds that modulate neurite growth of human induced pluripotent stem cell-derived neurons

ABSTRACT Development of technology platforms to perform compound screens of human induced pluripotent stem cell (hiPSC)-derived neurons with relatively high throughput is essential to realize their potential for drug discovery. Here, we demonstrate the feasibility of high-throughput screening of hiPSC-derived neurons using a high-content, image-based approach focused on neurite growth, a process that is fundamental to formation of neural networks and nerve regeneration. From a collection of 4421 bioactive small molecules, we identified 108 hit compounds, including 37 approved drugs, that target molecules or pathways known to regulate neurite growth, as well as those not previously associated with this process. These data provide evidence that many pathways and targets known to play roles in neurite growth have similar activities in hiPSC-derived neurons that can be identified in an unbiased phenotypic screen. The data also suggest that hiPSC-derived neurons provide a useful system to study the mechanisms of action and off-target activities of the approved drugs identified as hits, leading to a better understanding of their clinical efficacy and toxicity, especially in the context of specific human genetic backgrounds. Finally, the hit set we report constitutes a sublibrary of approved drugs and tool compounds that modulate neurites. This sublibrary will be invaluable for phenotypic analyses and interrogation of hiPSC-based disease models as probes for defining phenotypic differences and cellular vulnerabilities in patient versus control cells, as well as for investigations of the molecular mechanisms underlying human neurite growth in development and maintenance of neuronal networks, and nerve regeneration. Summary: High-throughput, small molecule screening of hiPSC-derived neurons using a high-content, image-based approach focused on neurite growth identified hit compounds, including approved drugs, which target molecules or pathways known to regulate neurite growth.