Conventional 3D printing technologies typically rely on open‐loop, calibrate‐then‐print operation procedures. An alternative approach is adaptive 3D printing, which is a closed‐loop method that ...combines real‐time feedback control and direct ink writing of functional materials in order to fabricate devices on moving freeform surfaces. Here, it is demonstrated that the changes of states in the 3D printing workspace in terms of the geometries and motions of target surfaces can be perceived by an integrated robotic system aided by computer vision. A hybrid fabrication procedure combining 3D printing of electrical connects with automatic pick‐and‐placing of surface‐mounted electronic components yields functional electronic devices on a free‐moving human hand. Using this same approach, cell‐laden hydrogels are also printed on live mice, creating a model for future studies of wound‐healing diseases. This adaptive 3D printing method may lead to new forms of smart manufacturing technologies for directly printed wearable devices on the body and for advanced medical treatments.
An adaptive 3D printing system capable of printing functional conductive inks and bioinks on moving freeform surfaces yields wireless electronics on a moving human hand and cell‐laden biostructures on mice. The concept of printing functional and biological materials via an autonomous, adaptive 3D printing system suggests a new paradigm for printing on‐the‐fly wearable electronics on moving targets and for surgical applications.
Chimeric antigen receptor (CAR) T cell therapy represents the first U.S. Food and Drug Administration approved gene therapy and these engineered cells function with unprecedented efficacy in the ...treatment of refractory CD19 positive hematologic malignancies. CAR translation to solid tumors is also being actively investigated; however, efficacy to date has been variable due to tumor-evolved mechanisms that inhibit local immune cell activity. To bolster the potency of CAR-T cells, modulation of the immunosuppressive tumor microenvironment with immune-checkpoint blockade is a promising strategy. The impact of this approach on hematological malignancies is in its infancy, and in this review we discuss CAR-T cells and their synergy with immune-checkpoint blockade.
Contrary to the prevailing professional opinion of the past few decades, recent experimental and clinical data support the fact that protein replacement therapy by allogeneic blood and marrow ...transplantation is not limited to freely diffusible molecules such as enzymes, but also large structural proteins such as collagens. A prime example is the cross-correction of type VII collagen deficiency in generalised severe recessive dystrophic epidermolysis bullosa, in which blood and marrow transplantation can attenuate the mucocutaneous manifestations of the disease and improve patients' quality of life. Although allogeneic blood and marrow transplantation can improve the integrity of the skin and mucous membranes, today's accomplishments are only the first steps on the long pathway to cure. Future strategies will be built on the lessons learned from these first transplant studies.
Clinical application of umbilical cord blood (UCB) as a source of hematopoietic stem cells for transplantation is limited by low CD34+ cell dose, increased risk of graft failure, and slow ...hematopoietic recovery. While the cell dose limitation is partially mitigated by using two UCB units, larger-dosed single units would be preferable. We have evaluated the feasibility and safety of StemRegenin-1 (SR-1), an aryl hydrocarbon receptor antagonist that expands CD34+ cells, by placing one of the two units in expansion culture. SR-1 produced a 330-fold increase in CD34+ cells and led to engraftment in 17/17 patients at a median of 15 days for neutrophils and 49 days for platelets, significantly faster than in patients treated with unmanipulated UCB. Taken together, the marked expansion, absence of graft failure, and enhanced hematopoietic recovery support testing of SR-1 expansion as a stand-alone graft and suggest it may ameliorate a limitation of UCB transplant.
Display omitted
•SR-1 led to significant expansion of CD34+ HSPCs in culture•Seventeen patients with hematological malignancy received SR-1 expanded UCB•SR-1 expanded cells were co-infused with a second unexpanded UCB unit•SR-1 expansion improved neutrophil and platelet recovery compared to controls
Clinical testing of the aryl hydrocarbon antagonist StemRegenin-1 showed robust expansion of hematopoietic stem and progenitor cells and an adequate safety profile in the setting of double UCB transplant, supporting its further testing for safety and efficacy as a stand-alone graft after myeloablative conditioning.
Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. ...Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.
Genome editing represents a promising strategy for the therapeutic correction of COL7A1 mutations that cause recessive dystrophic epidermolysis bullosa (RDEB). DNA cleavage followed by ...homology-directed repair (HDR) using an exogenous template has previously been used to correct COL7A1 mutations. HDR rates can be modest, and the double-strand DNA breaks that initiate HDR commonly result in accompanying undesired insertions and deletions (indels). To overcome these limitations, we applied an A•T→G•C adenine base editor (ABE) to correct two different COL7A1 mutations in primary fibroblasts derived from RDEB patients. ABE enabled higher COL7A1 correction efficiencies than previously reported HDR efforts. Moreover, ABE obviated the need for a repair template, and minimal indels or editing at off-target sites was detected. Base editing restored the endogenous type VII collagen expression and function in vitro. We also treated induced pluripotent stem cells (iPSCs) derived from RDEB fibroblasts with ABE. The edited iPSCs were differentiated into mesenchymal stromal cells, a cell population with therapeutic potential for RDEB. In a mouse teratoma model, the skin derived from ABE-treated iPSCs showed the proper deposition of C7 at the dermal–epidermal junction in vivo. These demonstrate that base editing provides an efficient and precise genome editing method for autologous cell engineering for RDEB.
This work describes a robust, simple method for generating cerebral organoids from human induced pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture ...medium. This method greatly facilitates access to cerebral organoid technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.
Tissue organoids are a promising technology that may accelerate development of the societal and NIH mandate for precision medicine. Here we describe a robust and simple method for generating cerebral organoids (cOrgs) from human pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. By using no additional neural induction components, cOrgs appeared on the hydrogel surface within 10–14 days, and under static culture conditions, they attained sizes up to 3 mm in greatest dimension by day 28. Histologically, the organoids showed neural rosette and neural tube‐like structures and evidence of early corticogenesis. Immunostaining and quantitative reverse‐transcription polymerase chain reaction demonstrated protein and gene expression representative of forebrain, midbrain, and hindbrain development. Physiologic studies showed responses to glutamate and depolarization in many cells, consistent with neural behavior. The method of cerebral organoid generation described here facilitates access to this technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.
Significance
Tissue organoids are a promising technology with many potential applications, such as pharmaceutical screens and development of in vitro disease models, particularly for human polygenic conditions where animal models are insufficient. This work describes a robust and simple method for generating cerebral organoids from human induced pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. This method, by virtue of its simplicity and use of defined materials, greatly facilitates access to cerebral organoid technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.
Cerebral adrenoleukodystrophy (cALD) remains a devastating neurodegenerative disease; only allogeneic hematopoietic cell transplantation (HCT) has been shown to provide long-term disease ...stabilization and survival. Sixty boys undergoing HCT for cALD from 2000 to 2009 were analyzed. The median age at HCT was 8.7 years; conditioning regimens and allograft sources varied. At HCT, 50% demonstrated a Loes radiographic severity score ≥ 10, and 62% showed clinical evidence of neurologic dysfunction. A total of 78% (n = 47) are alive at a median 3.7 years after HCT. The estimate of 5-year survival for boys with Loes score < 10 at HCT was 89%, whereas that for boys with Loes score ≥ 10 was 60% (P = .03). The 5-year survival estimate for boys absent of clinical cerebral disease at HCT was 91%, whereas that for boys with neurologic dysfunction was 66% (P = .08). The cumulative incidence of transplantation-related mortality at day 100 was 8%. Post-transplantation progression of neurologic dysfunction depended significantly on the pre-HCT Loes score and clinical neurologic status. We describe the largest single-institution analysis of survival and neurologic function outcomes after HCT in cALD. These trials were registered at www.clinicaltrials.gov as #NCT00176904, #NCT00668564, and #NCT00383448.
Present adoptive immunotherapy strategies are based on the re-targeting of autologous T-cells to recognize tumor antigens. As T-cell properties may vary significantly between patients, this approach ...can result in significant variability in cell potency that may affect therapeutic outcome. More consistent results could be achieved by generating allogeneic cells from healthy donors. An impediment to such an approach is the endogenous T-cell receptors present on T-cells, which have the potential to direct dangerous off-tumor antihost reactivity. To address these limitations, we assessed the ability of three different TCR-α-targeted nucleases to disrupt T-cell receptor expression in primary human T-cells. We optimized the conditions for the delivery of each reagent and assessed off-target cleavage. The megaTAL and CRISPR/Cas9 reagents exhibited the highest disruption efficiency combined with low levels of toxicity and off-target cleavage, and we used them for a translatable manufacturing process to produce safe cellular substrates for next-generation immunotherapies.
PDF
