(i9) United States (12) Patent Application Publication Healy et al. US 20070026518A1 (io) Pub. No... more (i9) United States (12) Patent Application Publication Healy et al. US 20070026518A1 (io) Pub. No.: US 2007/0026518 Al (43) Pub. Date: Feb. 1, 2007 (54) CONTROLLING STEM CELL DESTINY WITH TUNABLE MATRICES (75) Inventors: Kevin E. Healy, Moraga, CA (US); Beth Irwin, San Francisco, CA (US); Jacob Freas Pollock, Kensington, CA (US); David Schaffer, Pleasant Hill, CA (US); Krishanu Saha, Berkeley, CA (US); Ying Li, San Francisco, CA (US); Samuel Thomas Wall, San Francisco, CA (US) Correspondence Address: MORGAN, LEWIS & BOCKIUS ...
Human embryonic stem (ES) and induced pluripotent stem (iPS) cells survive and self-renew poorly ... more Human embryonic stem (ES) and induced pluripotent stem (iPS) cells survive and self-renew poorly when dissociated into single cells, making the isolation of genetically modified hES and hiPS cells a cumbersome process. We hypothesized that substrate-mediated signaling influences clonal growth and could be engineered to enhance this process. To this end, a library of cell-compatible protein-coated polymer substrates were utilized in a miniaturized, high-throughput nanoliter format. These nanoliter polymer substrate arrays were then screened for colony growth from a single hES cell. After screening thousands of protein-coated polymers, multiple hits' indicated that a specific range of polymeric structures could promote single cell growth. Propagation of hES cells on these substrates showed that the resulting colonies retained a pluripotent phenotype, even after subsequent passaging. These hits also were utilized with hiPS cells. By identifying substrates which support clonal growt...
Currently, new cellular models of human disease are emerging through the use of reprogramming tec... more Currently, new cellular models of human disease are emerging through the use of reprogramming technology. Direct reprogramming of human somatic cells to embryonic-like induced pluripotent stem (iPS) cells uses readily accessible skin, fat, blood, and hair cells. A primary use of such technology is to reprogram somatic cells from patients who are classified into a disease group, thus creating iPS cell lines popularly labeled as diseases in a dish. The disease-in-a-dish agenda thus frames disease as a phenomenon to be seen at the cellular level by laboratory scientists. This work will examine the assumptions built into diseases in a dish and further develop numerical models associated with this engineering endeavor. Applications in regenerative medicine, drug discovery, and disease modeling will be thoroughly discussed.
The present disclosure provides a device and a cell culture system comprising a substrate that ge... more The present disclosure provides a device and a cell culture system comprising a substrate that generates significant chemical ion signatures adapted for culturing stem cells. This disclosure further provides unique surface properties, such as surface wettability, along with defined polymer microspot environments in an array, for effectively supporting the propagation and differentiation of human pluripotent stem cells in vitro. Methods of culturing, maintenance, differentiating stem cells as well as reprogramming somatic cells into stem ...
Highly-regulated signals surrounding stem cells, such as growth factor signals and matrix mechani... more Highly-regulated signals surrounding stem cells, such as growth factor signals and matrix mechanical stiffness, have been implicated in modulating stem cell proliferation and maturation. However, tight control of proliferation and lineage commitment signals is rarely achieved during growth outside the body, since the spectrum of biochemical and mechanical signals that govern stem cell self-renewal and maturation are not fully understood. Therefore, stem cell control can potentially be enhanced through the ...
Stem cells are defined by their capacities for self-renewal and differentiation into one or more ... more Stem cells are defined by their capacities for self-renewal and differentiation into one or more cell lineages. Without tight regulation or control of these properties; however, any derivative cell population will exhibit a range of heterogeneous phenotypes, yielding artifacts that may complicate the development of pharmaceuticals and cell therapies. Recent work demonstrates that biomaterials (ie, matrices, scaffolds, culture substrates) can present key regulatory signals that combine with other environmental and genetic influences to ...
(i9) United States (12) Patent Application Publication Healy et al. US 20070026518A1 (io) Pub. No... more (i9) United States (12) Patent Application Publication Healy et al. US 20070026518A1 (io) Pub. No.: US 2007/0026518 Al (43) Pub. Date: Feb. 1, 2007 (54) CONTROLLING STEM CELL DESTINY WITH TUNABLE MATRICES (75) Inventors: Kevin E. Healy, Moraga, CA (US); Beth Irwin, San Francisco, CA (US); Jacob Freas Pollock, Kensington, CA (US); David Schaffer, Pleasant Hill, CA (US); Krishanu Saha, Berkeley, CA (US); Ying Li, San Francisco, CA (US); Samuel Thomas Wall, San Francisco, CA (US) Correspondence Address: MORGAN, LEWIS & BOCKIUS ...
SUMMARY Substrate composition significantly impacts human pluripotent stem cell (hPSC) self-renew... more SUMMARY Substrate composition significantly impacts human pluripotent stem cell (hPSC) self-renewal and differentiation, but relatively little is known about the role of endogenously produced extracellular matrix (ECM) components in regulating hPSC fates. Here we identify a-5 laminin as a signature ECM component endogenously synthesized by undifferentiated hPSCs cultured on defined substrates. Inducible shRNA knockdown and Cas9-mediated disruption of the LAMA5 gene dramatically reduced hPSC self-renewal and increased apoptosis without affecting the expression of pluripotency markers. Increased self-renewal and survival was restored to wild-type levels by culturing the LAMA5-deficient cells on exogenous laminin-521. Furthermore, treatment of LAMA5-deficient cells with blebbistatin or a ROCK in-hibitor partially restored self-renewal and diminished apoptosis. These results demonstrate that endogenous a-5 laminin promotes hPSC self-renewal in an autocrine and paracrine manner. This finding has implications for understanding how stem cells dynamically regulate their microenvironment to promote self-renewal and provides guidance for efforts to design substrates for stem cell bioprocessing.
A fundamental concern in disease modeling with stem-cell-derived cells is the need to connect inf... more A fundamental concern in disease modeling with stem-cell-derived cells is the need to connect information about cellular and tissue dysfunction in the lab with the manifestation of pathologies in individuals. To do this precisely necessitates linking personally identifiable information about individual patients, including medical records, biosensor data, and other potentially sensitive personal health information (PHI), to stem cell lines made from their donated tissue. Linking rich phenotypic data with stem cell lines, however, comes with a risk of informational harm, should donor anonymity be breached.
Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) can be achieved... more Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. Here we demonstrate that reprogramming is a continuous stochastic process where almost all donor cells eventually give rise to iPSCs upon continued growth and transcription factor expression. Additional inhibition the p53/p21 pathway or overexpression of Lin28 increased the cell division rate and resulted in an accelerated kinetics of iPSC formation that was directly proportional to the increase in cell proliferation. In contrast, Nanog overexpression accelerated reprogramming in a predominantly cell division rate independent manner. Quantitative analyses define distinct cell division rate dependent and independent modes for accelerating the stochastic course of reprogramming, and suggest that the number of cell divisions is a key parameter driving epigenetic reprogramming to pluripotency.
Proposed rules to protect research subjects will impede progress, say Krishanu Saha and J. Benjam... more Proposed rules to protect research subjects will impede progress, say Krishanu Saha and J. Benjamin Hurlbut. Instead, give donors more say in how samples are used.
Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have ve... more Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties, and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by ectopic induction of Oct4, Klf4, and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3beta (GSK3beta) and mitogen-activated protein kinase (ERK1/2) pathway. Forskolin, a protein kinase A pathway agonist which can induce Klf4 and Klf2 expression, transiently substitutes for the requirement for ectopic transgene expression. In contrast to conventional human ESCs, these epigenetically converted cells have growth properties, an X-chromosome activation state (XaXa), a gene expression profile, and a signaling pathway dependence that are highly similar to those of mouse ESCs. Finally, the same growth conditions allow the derivation of human induced pluripotent stem (iPS) cells with similar properties as mouse iPS cells. The generation of validated "naïve" human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans and may open up new opportunities for patient-specific, disease-relevant research.
(i9) United States (12) Patent Application Publication Healy et al. US 20070026518A1 (io) Pub. No... more (i9) United States (12) Patent Application Publication Healy et al. US 20070026518A1 (io) Pub. No.: US 2007/0026518 Al (43) Pub. Date: Feb. 1, 2007 (54) CONTROLLING STEM CELL DESTINY WITH TUNABLE MATRICES (75) Inventors: Kevin E. Healy, Moraga, CA (US); Beth Irwin, San Francisco, CA (US); Jacob Freas Pollock, Kensington, CA (US); David Schaffer, Pleasant Hill, CA (US); Krishanu Saha, Berkeley, CA (US); Ying Li, San Francisco, CA (US); Samuel Thomas Wall, San Francisco, CA (US) Correspondence Address: MORGAN, LEWIS & BOCKIUS ...
Human embryonic stem (ES) and induced pluripotent stem (iPS) cells survive and self-renew poorly ... more Human embryonic stem (ES) and induced pluripotent stem (iPS) cells survive and self-renew poorly when dissociated into single cells, making the isolation of genetically modified hES and hiPS cells a cumbersome process. We hypothesized that substrate-mediated signaling influences clonal growth and could be engineered to enhance this process. To this end, a library of cell-compatible protein-coated polymer substrates were utilized in a miniaturized, high-throughput nanoliter format. These nanoliter polymer substrate arrays were then screened for colony growth from a single hES cell. After screening thousands of protein-coated polymers, multiple hits' indicated that a specific range of polymeric structures could promote single cell growth. Propagation of hES cells on these substrates showed that the resulting colonies retained a pluripotent phenotype, even after subsequent passaging. These hits also were utilized with hiPS cells. By identifying substrates which support clonal growt...
Currently, new cellular models of human disease are emerging through the use of reprogramming tec... more Currently, new cellular models of human disease are emerging through the use of reprogramming technology. Direct reprogramming of human somatic cells to embryonic-like induced pluripotent stem (iPS) cells uses readily accessible skin, fat, blood, and hair cells. A primary use of such technology is to reprogram somatic cells from patients who are classified into a disease group, thus creating iPS cell lines popularly labeled as diseases in a dish. The disease-in-a-dish agenda thus frames disease as a phenomenon to be seen at the cellular level by laboratory scientists. This work will examine the assumptions built into diseases in a dish and further develop numerical models associated with this engineering endeavor. Applications in regenerative medicine, drug discovery, and disease modeling will be thoroughly discussed.
The present disclosure provides a device and a cell culture system comprising a substrate that ge... more The present disclosure provides a device and a cell culture system comprising a substrate that generates significant chemical ion signatures adapted for culturing stem cells. This disclosure further provides unique surface properties, such as surface wettability, along with defined polymer microspot environments in an array, for effectively supporting the propagation and differentiation of human pluripotent stem cells in vitro. Methods of culturing, maintenance, differentiating stem cells as well as reprogramming somatic cells into stem ...
Highly-regulated signals surrounding stem cells, such as growth factor signals and matrix mechani... more Highly-regulated signals surrounding stem cells, such as growth factor signals and matrix mechanical stiffness, have been implicated in modulating stem cell proliferation and maturation. However, tight control of proliferation and lineage commitment signals is rarely achieved during growth outside the body, since the spectrum of biochemical and mechanical signals that govern stem cell self-renewal and maturation are not fully understood. Therefore, stem cell control can potentially be enhanced through the ...
Stem cells are defined by their capacities for self-renewal and differentiation into one or more ... more Stem cells are defined by their capacities for self-renewal and differentiation into one or more cell lineages. Without tight regulation or control of these properties; however, any derivative cell population will exhibit a range of heterogeneous phenotypes, yielding artifacts that may complicate the development of pharmaceuticals and cell therapies. Recent work demonstrates that biomaterials (ie, matrices, scaffolds, culture substrates) can present key regulatory signals that combine with other environmental and genetic influences to ...
(i9) United States (12) Patent Application Publication Healy et al. US 20070026518A1 (io) Pub. No... more (i9) United States (12) Patent Application Publication Healy et al. US 20070026518A1 (io) Pub. No.: US 2007/0026518 Al (43) Pub. Date: Feb. 1, 2007 (54) CONTROLLING STEM CELL DESTINY WITH TUNABLE MATRICES (75) Inventors: Kevin E. Healy, Moraga, CA (US); Beth Irwin, San Francisco, CA (US); Jacob Freas Pollock, Kensington, CA (US); David Schaffer, Pleasant Hill, CA (US); Krishanu Saha, Berkeley, CA (US); Ying Li, San Francisco, CA (US); Samuel Thomas Wall, San Francisco, CA (US) Correspondence Address: MORGAN, LEWIS & BOCKIUS ...
SUMMARY Substrate composition significantly impacts human pluripotent stem cell (hPSC) self-renew... more SUMMARY Substrate composition significantly impacts human pluripotent stem cell (hPSC) self-renewal and differentiation, but relatively little is known about the role of endogenously produced extracellular matrix (ECM) components in regulating hPSC fates. Here we identify a-5 laminin as a signature ECM component endogenously synthesized by undifferentiated hPSCs cultured on defined substrates. Inducible shRNA knockdown and Cas9-mediated disruption of the LAMA5 gene dramatically reduced hPSC self-renewal and increased apoptosis without affecting the expression of pluripotency markers. Increased self-renewal and survival was restored to wild-type levels by culturing the LAMA5-deficient cells on exogenous laminin-521. Furthermore, treatment of LAMA5-deficient cells with blebbistatin or a ROCK in-hibitor partially restored self-renewal and diminished apoptosis. These results demonstrate that endogenous a-5 laminin promotes hPSC self-renewal in an autocrine and paracrine manner. This finding has implications for understanding how stem cells dynamically regulate their microenvironment to promote self-renewal and provides guidance for efforts to design substrates for stem cell bioprocessing.
A fundamental concern in disease modeling with stem-cell-derived cells is the need to connect inf... more A fundamental concern in disease modeling with stem-cell-derived cells is the need to connect information about cellular and tissue dysfunction in the lab with the manifestation of pathologies in individuals. To do this precisely necessitates linking personally identifiable information about individual patients, including medical records, biosensor data, and other potentially sensitive personal health information (PHI), to stem cell lines made from their donated tissue. Linking rich phenotypic data with stem cell lines, however, comes with a risk of informational harm, should donor anonymity be breached.
Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) can be achieved... more Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. Here we demonstrate that reprogramming is a continuous stochastic process where almost all donor cells eventually give rise to iPSCs upon continued growth and transcription factor expression. Additional inhibition the p53/p21 pathway or overexpression of Lin28 increased the cell division rate and resulted in an accelerated kinetics of iPSC formation that was directly proportional to the increase in cell proliferation. In contrast, Nanog overexpression accelerated reprogramming in a predominantly cell division rate independent manner. Quantitative analyses define distinct cell division rate dependent and independent modes for accelerating the stochastic course of reprogramming, and suggest that the number of cell divisions is a key parameter driving epigenetic reprogramming to pluripotency.
Proposed rules to protect research subjects will impede progress, say Krishanu Saha and J. Benjam... more Proposed rules to protect research subjects will impede progress, say Krishanu Saha and J. Benjamin Hurlbut. Instead, give donors more say in how samples are used.
Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have ve... more Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties, and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by ectopic induction of Oct4, Klf4, and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3beta (GSK3beta) and mitogen-activated protein kinase (ERK1/2) pathway. Forskolin, a protein kinase A pathway agonist which can induce Klf4 and Klf2 expression, transiently substitutes for the requirement for ectopic transgene expression. In contrast to conventional human ESCs, these epigenetically converted cells have growth properties, an X-chromosome activation state (XaXa), a gene expression profile, and a signaling pathway dependence that are highly similar to those of mouse ESCs. Finally, the same growth conditions allow the derivation of human induced pluripotent stem (iPS) cells with similar properties as mouse iPS cells. The generation of validated "naïve" human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans and may open up new opportunities for patient-specific, disease-relevant research.
Many avenues exist for human pluripotent stem cells (hPSCs) to impact medical care, but they may ... more Many avenues exist for human pluripotent stem cells (hPSCs) to impact medical care, but they may have their greatest impact on the development of precision medicine. Recent advances in genome editing and stem cell technology have enabled construction of clinically-relevant, genotype-specific “disease-in-a-dish” models. In this review, we outline the use of genome-edited hPSCs in precision disease modeling and drug screening as well as describe methodological advances in scarless genome editing. Scarless genome-editing approaches are attractive for genotype-specific disease modeling as only the intended DNA base-pair edits are incorporated without additional genomic modification. Emerging evidentiary standards for development and approval of precision therapies are likely to increase application of disease models derived from genome-edited hPSCs.
Emerging manufacturing processes to generate regenerative advanced therapies can involve extensiv... more Emerging manufacturing processes to generate regenerative advanced therapies can involve extensive genomic and/or epigenomic manipulation of autologous or allogeneic cells. These cell engineering processes need to be carefully controlled and standardized to maximize safety and efficacy in clinical trials. Engineered biomaterials with smart and tunable properties offer an intriguing tool to provide or deliver cues to retain stemness, direct differentiation, promote reprogramming, manipulate the genome, or select functional phenotypes. This review discusses the use of engineered biomaterials to control human cell manufacturing. Future work exploiting engineered biomaterials has the potential to generate manufacturing processes that produce standardized cells with well-defined critical quality attributes appropriate for clinical testing.
Writing specific DNA sequences into the human genome is challenging with non-viral gene-editing r... more Writing specific DNA sequences into the human genome is challenging with non-viral gene-editing reagents, since most of the edited sequences contain various imprecise insertions or deletions. We developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to complex CRISPR-Cas9 ribonucleoproteins with a nucleic acid donor template, as well as other biotinylated molecules such as quantum dots. In human cells, tailored S1mplexes increase the ratio of precisely edited to imprecisely edited alleles up to 18-fold higher than standard gene-editing methods, and enrich cell populations containing multiplexed precise edits up to 42-fold. These advances with versatile, preassembled reagents could greatly reduce the time and cost of in vitro or ex vivo gene-editing applications in precision medicine and drug discovery and aid in the development of increased and serial dosing regimens for somatic gene editing in vivo.
Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, d... more Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a puromycin N-acetyl-transferase (PAC) gene, whose expression and translation is linked to that of Cas9, we transiently select for cells based on their early levels of Cas9 protein. Under optimized conditions, co-delivery with single-stranded donor DNA enabled isolation of clonal cell populations containing both heterozygous and homozygous precise genome edits in as little as 2 weeks without requiring cell sorting or high-throughput sequencing. Edited cells isolated using this method did not contain any detectable off-target mutations and displayed expected functional phenotypes after directed differentiation. We apply the approach to a variety of genomic loci in five hPSC lines cultured using both feeder and feeder-free conditions.
The next generation of therapeutic products to be approved for the clinic is anticipated to be ce... more The next generation of therapeutic products to be approved for the clinic is anticipated to be cell therapies, termed "living drugs" for their capacity to dynamically and temporally respond to changes during their production ex vivo and after their administration in vivo. Genetically engineered chimeric antigen receptor (CAR) T cells have rapidly developed into powerful tools to harness the power of immune system manipulation against cancer. Regulatory agencies are beginning to approve CAR T cell therapies due to their striking efficacy in treating some hematological malignancies. However, the engineering and manufacturing of such cells remains a challenge for widespread adoption of this technology. Bioengineering approaches including biomaterials, synthetic biology, metabolic engineering, process control and automation, and in vitro disease modeling could offer promising methods to overcome some of these challenges. Here, we describe the manufacturing process of CAR T cells, highlighting potential roles for bioengineers to partner with biologists and clinicians to advance the manufacture of these complex cellular products under rigorous regulatory and quality control.
Millions of people globally are at high risk for neurodegenerative disorders, infertility or havi... more Millions of people globally are at high risk for neurodegenerative disorders, infertility or having children with a disability as a result of the Fragile X (FX) premutation, a genetic abnormality in FMR1 that is underdiagnosed. Despite the high prevalence of the FX premutation and its effect on public health and family planning, most FX premutation carriers are unaware of their condition. Since genetic testing for the premutation is resource intensive, it is not practical to screen individuals for FX premutation status using genetic testing. In a novel approach to phenotyping, we have utilized audio recordings and cognitive profiling assessed via self-administered questionnaires on 200 females. Machine-learning methods were developed to discriminate FX premutation carriers from mothers of children with autism spectrum disorders, the comparison group. By using a random forest classifier, FX premutation carriers could be identified in an automated fashion with high precision and recall (0.81 F1 score). Linguistic and cognitive phenotypes that were highly associated with FX premutation carriers were high language dysfluency, poor ability to organize material, and low self-monitoring. Our framework sets the foundation for computational phenotyping strategies to pre-screen large populations for this genetic variant with nominal costs.
The driving forces for the (111) to (100) texture transformation often observed during annealing ... more The driving forces for the (111) to (100) texture transformation often observed during annealing of thin face-centered cubic metal films were investigated. Thin passivated silver films were produced with and without Ti adhesion layers. Stresses were measured in situ during heating to induce the texture transformation, and the texture was characterized using x-ray diffraction. Sufficiently thin films did not transform and sufficiently thick films transformed fully. Intermediate thickness films transformed to an extent dependent on thickness, leading to stable mixed textures. In the prevailing thermodynamic model, texture transformation is attributed to minimization of strain and interface energies. However, calculations using the measured stresses, known elastic constants, and estimated interface energies in this model reveal that the stresses are not sufficient to cause the texture transformation and, furthermore, that variations in interface energy cannot lead to the observed behavior. The results suggest that neither the interface energy nor the stress plays decisive roles in the texture transformation.
Forced expression of transcription factors epigenetically reprograms somatic cells harvested from... more Forced expression of transcription factors epigenetically reprograms somatic cells harvested from routine skin biopsies into induced pluripotent stem cells (iPSCs). Human iPSCs are key resources for drug discovery, regenerative medicine and tissue engineering. Here we developed a materials approach to explore how culture substrates could impact factor-mediated reprogramming of human fibroblasts. A materials library consisting of nanofibrous substrates with randomly oriented and aligned structures was prepared by electrospinning four polymers [polylactic acid (PLA), polycaprolactone (PCL), thermoplastic polyurethane (TPU) and polypropylene carbonate (PPC)] into nanofiber orientations. Adsorbing protein to each substrate permitted robust attachment of fibroblasts to all substrates. Fibroblasts on aligned substrates had elongated nuclei, but after reprogramming factor expression, nuclei became more circular. Reprogramming factors could override the nuclear shape constraints imposed by nanofibrous substrates, and the majority of substrates supported full reprogramming. Early culture on PCL and TPU substrates promoted reprogramming, and TGF-β repressed substrate effects. Partial least squares modeling of the biochemical and biophysical cues within our reprogramming system identified TGF-β and polymer identity as important cues governing cellular reprogramming responses. We believe that our approach of using a nanofibrous materials library can be used to dissect molecular mechanisms of reprogramming and generate novel substrates that enhance epigenetic reprogramming.
Many biological processes, including differentiation, reprogramming, and disease transformations,... more Many biological processes, including differentiation, reprogramming, and disease transformations, involve transitions of cells through distinct states. Direct, unbiased investigation of cell states and their transitions is challenging due to several factors, including limitations of single-cell assays. Here we present a stochastic model of cellular transitions that allows underlying single-cell information, including cell-state-specific parameters and rates governing transitions between states, to be estimated from genome-wide, population-averaged time-course data. The key novelty of our approach lies in specifying latent stochastic models at the single-cell level, and then aggregating these models to give a likelihood that links parameters at the single-cell level to observables at the population level. We apply our approach in the context of reprogramming to pluripotency. This yields new insights, including profiles of two intermediate cell states, that are supported by independent single-cell studies. Our model provides a general conceptual framework for the study of cell transitions, including epigenetic transformations.
Many biological processes, including differentiation, reprogramming, and disease transformations,... more Many biological processes, including differentiation, reprogramming, and disease transformations, involve transitions of cells through distinct states. Direct, unbiased investigation of cell states and their transitions is challenging due to several factors, including limitations of single-cell assays. Here we present a stochastic model of cellular transitions that allows underlying single-cell information, including cell-state-specific parameters and rates governing transitions between states, to be estimated from genome-wide, population-averaged time-course data. The key novelty of our approach lies in specifying latent stochastic models at the single-cell level, and then aggregating these models to give a likelihood that links parameters at the single-cell level to observables at the population level. We apply our approach in the context of reprogramming to pluripotency. This yields new insights, including profiles of two intermediate cell states, that are supported by independent single-cell studies. Our model provides a general conceptual framework for the study of cell transitions, including epigenetic transformations.
Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to co... more Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few
The driving forces for the (111) to (100) texture transformation often observed during annealing ... more The driving forces for the (111) to (100) texture transformation often observed during annealing of thin face-centered cubic metal films were investigated. Thin passivated silver films were produced with and without Ti adhesion layers. Stresses were measured in situ during heating to induce the texture transformation, and the texture was characterized using x-ray diffraction. Sufficiently thin films did not transform and sufficiently thick films transformed fully. Intermediate thickness films transformed to an extent dependent on thickness, leading to stable mixed textures. In the prevailing thermodynamic model, texture transformation is attributed to minimization of strain and interface energies. However, calculations using the measured stresses, known elastic constants, and estimated interface energies in this model reveal that the stresses are not sufficient to cause the texture transformation and, furthermore, that variations in interface energy cannot lead to the observed behavior. The results suggest that neither the interface energy nor the stress plays decisive roles in the texture transformation.
Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of... more Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of a fluorescent mCherry reporter transgene within human embryonic kidney cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression, 24 h after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery. Statement of Significance CRISPR-Cas9 is a new gene-editing technology for “genome surgery” that is anticipated to treat genetic diseases. This technology uses multiple components of the Cas9 system to cut out disease-causing mutations in the human genome and precisely suture in therapeutic sequences. Biomaterials based delivery strategies could help transition these technologies to the clinic. The design space for materials based delivery strategies is vast and optimization is essential to ensuring the safety and efficacy of these treatments. Therefore, new methods are required to rapidly and systematically screen gene-editing efficacy in human cells. This work utilizes an innovative platform to generate and screen many formulations of synthetic biomaterials and components of the CRISPR-Cas9 system in parallel. On this platform, we watch genome surgery in action using high content image analysis. These capabilities enabled us to identify formulation parameters for Cas9-material complexes that can optimize gene-editing in a specific human cell type.
Disclosed are methods for cell transfection and regulating cellular behavior. More particularly, ... more Disclosed are methods for cell transfection and regulating cellular behavior. More particularly, the present disclosure relates to methods of non-viral cell transfection and regulating cellular behavior using mineral coatings that allow for the enhanced transfection of cells with reduced cytotoxicity. The mineral coatings bind biomaterials and provide a source of calcium and phosphate ions to enhance transfection. The present disclosure also provides a high throughput platform for screening non-viral transfection of cells. The methods of the present disclosure also provide an advantageous biomaterial delivery platform because the mineral coatings may be deposited on various medical device materials after being specifically developed using the high throughput screening platform.
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