Rural parents' attitudes and beliefs on the COVID-19 pediatric vaccine: An explanatory study.

The coronavirus disease 2019 (COVID-19) first came to the Unites States in January 2020. Though adult and pediatric vaccines became available to the public, vaccine uptake among youth and particularly younger children has been gradual. This explanatory study aimed to better understand parents' attitudes and beliefs of the pediatric COVID-19 vaccine and the barriers and facilitators to vaccine uptake in a rural community through a brief, online demographic survey, and in-depth qualitative interviews. Forty-one in depth interviews were conducted with parents (31-English and 10-Spanish-speaking) residing in rural and frontier counties in Colorado between September 2021 and February 2022. Six emergent themes related to COVID-19 pediatric vaccine uptake were identified among the population. These themes spanned the three levels of influence in the Social Ecological Model (individual, interpersonal, and community levels). The six themes were identified as such; 1) Vaccine accessibility was associated with pediatric COVID vaccine uptake in rural communities, 2) Previous pediatric vaccine behaviors were not associated with COVID-19 pediatric vaccine uptake, 3) Perceived health status of a child or family member influenced pediatric COVID-19 vaccine uptake, 4) COVID-19 health seeking behaviors, like COVID pediatric vaccine uptake, are influenced by an individual's prosocial or individualistic perspectives, 5) Child autonomy and "age of consent" frames vaccine decision making behaviors in parents, and lastly 6) Social networks impacted COVID-19 pediatric vaccine decision making. These findings inform next steps for COVID-19 pediatric vaccine uptake including targeted and tailored messaging for communities (cues to actions), engaging youth stakeholders, and identifying trusted sources to build rapport and trust between health professionals and community members. The growing vaccine hesitancy among parents has serious implications for disease eradication and future viral outbreaks. Understanding the perceived barriers and facilitators to pediatric vaccine uptake is important to maintain the health of our youth and communities.

Enhanced phagemid particle gene transfer in camptothecin-treated carcinoma cells.

Engineered phage-based vectors are an attractive alternative strategy for gene delivery because they possess no natural mammalian cell tropism and can be genetically modified for specific applications. Genotoxic treatments that increase the transduction efficiency of single-stranded adeno-associated virus were tested on cells transfected by single-stranded phage. Indeed, green fluorescent protein transgene expression by epidermal growth factor-targeted phagemid particles increased with heat shock, UV irradiation, and camptothecin (CPT) treatment. CPT resulted in transduction efficiencies of 30-45% in certain human carcinoma cell lines and reduced the minimal dose needed to detect green fluorescent protein-expressing cells to as low as 1-10 particles/cell. Targeted phage transduction was effective in many tumor cell lines and in prostate tumor xenografts with CPT treatment. Taken together, these data suggest the feasibility of using phage-based vectors for therapeutic gene delivery to cancer cells.

A novel lineage restricted, pericyte-like cell line isolated from human embryonic stem cells.

Pericytes (PCs) are endothelium-associated cells that play an important role in normal vascular function and maintenance. We developed a method comparable to GMP quality protocols for deriving self-renewing perivascular progenitors from the human embryonic stem cell (hESC), line ESI-017. We identified a highly scalable, perivascular progenitor cell line that we termed PC-A, which expressed surface markers common to mesenchymal stromal cells. PC-A cells were not osteogenic or adipogenic under standard differentiation conditions and showed minimal angiogenic support function in vitro. PC-A cells were capable of further differentiation to perivascular progenitors with limited differentiation capacity, having osteogenic potential (PC-O) or angiogenic support function (PC-M), while lacking adipogenic potential. Importantly, PC-M cells expressed surface markers associated with pericytes. Moreover, PC-M cells had pericyte-like functionality being capable of co-localizing with human umbilical vein endothelial cells (HUVECs) and enhancing tube stability up to 6 days in vitro. We have thus identified a self-renewing perivascular progenitor cell line that lacks osteogenic, adipogenic and angiogenic potential but is capable of differentiation toward progenitor cell lines with either osteogenic potential or pericyte-like angiogenic function. The hESC-derived perivascular progenitors described here have potential applications in vascular research, drug development and cell therapy.

Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling.

Adult Versus Pluripotent Stem Cell-Derived Mesenchymal Stem Cells: The Need for More Precise Nomenclature.

The complexity of human pluripotent stem cell (hPSC) fate represents both opportunity and challenge. In theory, all somatic cell types can be differentiated from hPSCs, opening the door to many opportunities in transplant medicine. However, such clinical applications require high standards of purity and identity, that challenge many existing protocols. This underscores the need for increasing precision in the description of cell identity during hPSC differentiation. We highlight one salient example, namely, the numerous published reports of hPSC-derived mesenchymal stem cells (MSCs). We suggest that many of these reports likely represent an improper use of certain cluster of differentiation (CD) antigens in defining bone marrow-derived MSCs. Instead, most such hPSC-derived mesenchymal cells are likely a complex mixture of embryonic anlagen, primarily of diverse mesodermal and neural crest origins, making precise identification, reproducible manufacture, and uniform differentiation difficult to achieve. We describe a potential path forward that may provide more precision in nomenclature, and cells with higher purity and identity for potential therapeutic use.

The germline/soma dichotomy: implications for aging and degenerative disease.

Human somatic cells are mortal due in large part to telomere shortening associated with cell division. Limited proliferative capacity may, in turn, limit response to injury and may play an important role in the etiology of age-related pathology. Pluripotent stem cells cultured in vitro appear to maintain long telomere length through relatively high levels of telomerase activity. We propose that the induced reversal of cell aging by transcriptional reprogramming, or alternatively, human embryonic stem cells engineered to escape immune surveillance, are effective platforms for the industrial-scale manufacture of young cells for the treatment of age-related pathologies. Such cell-based regenerative therapies will require newer manufacturing and delivery technologies to insure highly pure, identified and potent pluripotency-based therapeutic formulations.

Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection.

Phage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature. However, the selection of natural ligands using phage display has been limited because of significant problems associated with the display of complex cDNA repertoires. Here we describe the use of cDNA fragmentation and open reading frame (ORF) selection to display a human placental cDNA library on the pIII coat protein of filamentous phage. The library was enriched for ORFs by selecting cDNA-beta-lactamase fusion proteins on ampicillin, resulting in a cDNA population having 97% ORFs. The ORF-selected cDNAs were fused to pIII in the phagemid vector, pUCMG4CT-198, and the library was rescued with a pIII-deleted helper phage for multivalent display. The resulting phagemid particle library consisted of 87% ORFs, compared to only 6% ORFs when prepared without ORF selection. Western blot analysis indicated cDNA-pIII fusion protein expression in eight out of nine ORF clones tested, and seven of the ORF encoded peptides were displayed multivalently. The high level of cDNA expression obtained by ORF selection suggests that ORF-enriched phage cDNA libraries prepared by these methods will be useful as functional genomics tools for identifying natural ligands from various source tissues.

Evolving phage vectors for cell targeted gene delivery.

We adapted filamentous phage vectors for targeted gene delivery to mammalian cells by inserting a mammalian reporter gene expression cassette (GFP) into the vector backbone and fusing the pIII coat protein to a cell targeting ligand (i.e. FGF2, EGF). Like transfection with animal viral vectors, targeted phage gene delivery is concentration, time, and ligand dependent. Importantly, targeted phage particles are specific for the appropriate target cell surface receptor. Phage have distinct advantages over existing gene therapy vectors because they are simple, economical to produce at high titer, have no intrinsic tropism for mammalian cells, and are relatively simple to genetically modify and evolve. Initially transduction by targeted phage particles was low resulting in foreign gene expression in 1-2% of transfected cells. We increased transduction efficiency by modifying both the transfection protocol and vector design. For example, we stabilized the display of the targeting ligand to create multivalent phagemid-based vectors with transduction efficiencies of up to 45% in certain cell lines when combined with genotoxic treatment. Taken together, these studies establish that the efficiency of phage-mediated gene transfer can be significantly improved through genetic modification. We are currently evolving phage vectors with enhanced cell targeting, increased stability, reduced immunogenicity and other properties suitable for gene therapy.

Selection of internalizing ligand-display phage using rolling circle amplification for phage recovery.

Selection of phage libraries against complex living targets such as whole cells or organs can yield valuable targeting ligands without prior knowledge of the targeted receptor. Our previous studies have shown that noninfective multivalent ligand display phagemids internalize into mammalian cells more efficiently than their monovalent counterparts suggesting that cell-based selection of internalizing ligands might be improved using multivalently displayed peptides, antibodies or cDNAs. However, alternative methods of phage recovery are needed to select phage from noninfective libraries. To this end, we reasoned that rolling circle amplification (RCA) of phage DNA could be used to recover noninfective phage. In feasibility studies, we obtained up to 1.5 million-fold enrichment of internalizing EGF-targeted phage using RCA. When RCA was applied to a large random peptide library, eight distinct human prostate carcinoma cell-internalizing peptides were isolated within three selection rounds. These data establish RCA as an alternative to infection for phage recovery that can be used to identify peptides from noninfective phage display libraries or infective libraries under conditions where there is the potential for loss of phage infectivity.

Human embryonic stem cell-derived neural crest cells capable of expressing markers of osteochondral or meningeal-choroid plexus differentiation.

AIMS: The transcriptome and fate potential of three diverse human embryonic stem cell-derived clonal embryonic progenitor cell lines with markers of cephalic neural crest are compared when differentiated in the presence of combinations of TGFß3, BMP4, SCF and HyStem-C matrices. MATERIALS & METHODS: The cell lines E69 and T42 were compared with MEL2, using gene expression microarrays, immunocytochemistry and ELISA. RESULTS: In the undifferentiated progenitor state, each line displayed unique markers of cranial neural crest including TFAP2A and CD24; however, none expressed distal HOX genes including HOXA2 or HOXB2, or the mesenchymal stem cell marker CD74. The lines also showed diverse responses when differentiated in the presence of exogenous BMP4, BMP4 and TGFß3, SCF, and SCF and TGFß3. The clones E69 and T42 showed a profound capacity for expression of endochondral ossification markers when differentiated in the presence of BMP4 and TGFß3, choroid plexus markers in the presence of BMP4 alone, and leptomeningeal markers when differentiated in SCF without TGFß3. CONCLUSION: The clones E69 and T42 may represent a scalable source of primitive cranial neural crest cells useful in the study of cranial embryology, and potentially cell-based therapy.

Identification of human embryonic progenitor cell targeting peptides using phage display.

Human pluripotent stem (hPS) cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS) cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES) cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken together these data suggest that selection of phage display libraries against a clonal progenitor stem cell population can be used to identify progenitor stem cell targeting peptides. The peptides may be useful for monitoring hPS cell differentiation and for the development of cell enrichment procedures to improve the efficiency of directed differentiation toward clinically relevant human cell types.

The ACTCellerate initiative: large-scale combinatorial cloning of novel human embryonic stem cell derivatives.

Human embryonic stem cells offer a scalable and renewable source of all somatic cell types. Human embryonic progenitor (hEP) cells are partially differentiated endodermal, mesodermal and ectodermal cell types that have not undergone terminal differentiation and express an embryonic pattern of gene expression. Here, we describe a large-scale and reproducible method of isolating a diverse library of clonally purified hEP cell lines, many of which are capable of extended propagation in vitro. Initial microarray and non-negative matrix factorization gene-expression profiling suggests that the library consists of at least 140 distinct clones and contains many previously uncharacterized cell types derived from all germ layers that display diverse embryo- and site-specific homeobox gene expression. Despite the expression of many oncofetal genes, none of the hEP cell lines tested led to tumor formation when transplanted into immunocompromised mice. All hEP lines studied appear to have a finite replicative lifespan but have longer telomeres than most fetal- or adult-derived cells, thereby facilitating their use in the manufacture of purified lineages for research and human therapy.