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Beta‐thalassemia is one of the most common recessive genetic diseases, caused by mutations in the HBB gene. Over 200 different types of mutations in the HBB gene containing three exons have been identified in patients with β‐thalassemia (β‐thal) whereas a homozygous mutation in exon 1 causes sickle cell disease (SCD). Novel therapeutic strategies to permanently correct the HBB mutation in stem cells that are able to expand and differentiate into erythrocytes producing corrected HBB proteins are highly desirable. Genome editing aided by CRISPR/Cas9 and other site‐specific engineered nucleases offers promise to precisely correct a genetic mutation in the native genome without alterations in other parts of the human genome. Although making a sequence‐specific nuclease to enhance correction of a specific HBB mutation by homology‐directed repair (HDR) is becoming straightforward, targeting various HBB mutations of β‐thal is still challenging because individual guide RNA as well as a donor DNA template for HDR of each type of HBB gene mutation have to be selected and validated. Using human induced pluripotent stem cells (iPSCs) from two β‐thal patients with different HBB gene mutations, we devised and tested a universal strategy to achieve targeted insertion of the HBB cDNA in exon 1 of HBB gene using Cas9 and two validated guide RNAs. We observed that HBB protein production was restored in erythrocytes derived from iPSCs of two patients. This strategy of restoring functional HBB gene expression will be able to correct most types of HBB gene mutations in β‐thal and SCD. Stem Cells Translational Medicine 2018;7:87–97 A universal strategy to repair various mutations of the HBB gene causing β‐thalassemia and sickle cell disease. Authors use Clustered Regularly Interspaced Short Palindromic Repeats‐mediated genome editing for targeted insertion of a functional HBB cDNA together with a GFP marker gene. After corrected human induced pluripotent stem cell lines are obtained, they differentiate them to erythrocytes that produce HBB proteins as well as GFP heterologous proteins.

Diabetes is a risk factor for worse outcomes following acute myocardial infarction (AMI). In this study, we tested the hypothesis that SDF‐1:CXCR4 expression is compromised in post‐AMI in diabetes, and that reversal of this defect can reverse the adverse effects of diabetes. Mesenchymal stem cells (MSC) isolated from green fluorescent protein (GFP) transgenic mice (control MSC) were induced to overexpress stromal cell‐derived factor‐1 (SDF‐1). SDF‐1 expression in control MSC and SDF‐1‐overexpressing MSC (SDF‐1:MSC) were quantified using enzyme‐linked immunosorbent assay (ELISA). AMI was induced on db/db and control mice. Mice were randomly selected to receive infusion of control MSC, SDF‐1:MSC, or saline into the border zone after AMI. Serial echocardiography was used to assess cardiac function. SDF‐1 and CXCR4 mRNA expression in the infarct zone of db/db mice and control mice were quantified. Compared to control mice, SDF‐1 levels were decreased 82%, 91%, and 45% at baseline, 1 day and 3 days post‐AMI in db/db mice, respectively. CXCR4 levels are increased 233% at baseline and 54% 5 days post‐AMI in db/db mice. Administration of control MSC led to a significant improvement in ejection fraction (EF) in control mice but not in db/db mice 21 days after AMI. In contrast, administration of SDF‐1:MSC produced a significant improvement in EF in both control mice and db/db mice 21 days after AMI. The SDF‐1:CXCR4 axis is compromised in diabetes, which appears to augment the deleterious consequences of AMI. Over‐express of SDF‐1 expression in diabetes rescues cardiac function post AMI. Our results suggest that modulation of SDF‐1 may improve post‐AMI cardiac repair in diabetes. Stem Cells Translational Medicine 2018;7:115–124 The SDF‐1:CXCR4 axis is compromised in diabetes, which appears to augment the deleterious consequences of acute myocardial infarction (AMI). Over‐express of stromal cell‐derived factor‐1 (SDF‐1) expression in diabetes rescues cardiac function post AMI. Results suggest that modulation of SDF‐1 may improve post‐AMI cardiac repair in diabetes.

Stem cells from human corneal stroma (CSSC) suppress corneal stromal scarring in a mouse wound‐healing model and promote regeneration of native transparent tissue (PMID:25504883). This study investigated efficacy of compressed collagen gel (CCG) as a vehicle to deliver CSSC for corneal therapy. CSSC isolated from limbal stroma of human donor corneas were embedded in soluble rat‐tendon collagen, gelled at 37°C, and partially dehydrated to a thickness of 100 µm by passive absorption. The CCG disks were dimensionally stable, easy to handle, and could be adhered securely to de‐epithelialized mouse cornea with fibrin‐based adhesive. CSSC in CCG maintained >80% viability for >1 week in culture media and could be cryopreserved in 20% fetal bovine serum‐10%DMSO in liquid nitrogen. CCG containing as few as 500 CSSC effectively prevented visible scarring and suppressed expression of fibrotic Col3a1 mRNA. CSSC in CCG were more effective at blocking scarring on a per‐cell basis than CSSC delivered directly in a fibrin gel as previously described. Collagen‐embedded cells retained the ability to suppress corneal scarring after conventional cryopreservation. This study demonstrates use of a common biomaterial that can facilitate storage and handling of stem cells in a manner that may provide off‐the‐shelf delivery of stem cells as a therapy for corneal scarring. Stem Cells Translational Medicine 2018;7:487–494 Corneal stromal stem cells (A) mixed with soluble collagen (B) are gelled in a 24‐well culture plate (C). Cells are concentrated by absorption with a fibrous plunger (D) forming a 100 μm thick disk (E). 2 mm disks punched with a trephine are cryopreserved (F) or adhered to the surface of a wounded cornea with fibrin adhesive (G) inducing corneal regeneration (H).

Mesenchymal stem cells from corneal stromal stem cells (CSSC) prevent fibrotic scarring and stimulate regeneration of transparent stromal tissue after corneal wounding in mice. These effects rely on the ability of CSSC to block neutrophil infiltration into the damaged cornea. The current study investigated the hypothesis that tissue regeneration by CSSC is mediated by secreted extracellular vesicles (EVs). CSSC produced EVs 130–150 nm in diameter with surface proteins that include CD63, CD81, and CD9. EVs from CSSC reduced visual scarring in murine corneal wounds as effectively as did live cells, but EVs from human embryonic kidney (HEK)293T cells had no regenerative properties. CSSC EV treatment of wounds decreased expression of fibrotic genes Col3a1 and Acta2, blocked neutrophil infiltration, and restored normal tissue morphology. CSSC EVs labeled with carboxyfluorescein succinimidyl ester dye, rapidly fused with corneal epithelial and stromal cells in culture, transferring microRNA (miRNA) to the target cells. Knockdown of mRNA for Alix, a component of the endosomal sorting complex required for transport, using siRNA, resulted in an 85% reduction of miRNA in the secreted EVs. The EVs with reduced miRNA were ineffective at blocking corneal scarring. Furthermore, CSSC with reduced Alix expression also lost their regenerative function, suggesting EVs as an obligate component in the delivery of miRNA. The results of these studies support an essential role for extracellular vesicles in the process by which CSSC cells block scarring and initiate regeneration of transparent corneal tissue after wounding. EVs appear to serve as a delivery vehicle for miRNA, which affects the regenerative action. Stem Cells Translational Medicine 2019;8:1192–1201 Mesenchymal stem cells from human corneal stroma (CSSC) induce regeneration of transparent corneal tissue in wounded corneas (A). Extracellular vesicles secreted by CSSC exhibit the same ability (B). Knockdown of Alix protein in the CSSC cells results in loss of miRNA in the extracellular vesicles. These extracellular vesicles do not prevent corneal scarring (C).

The effects of mesenchymal stem cell (MSC)‐derived extracellular vesicles were compared with those of MSCs i.v. delivered 1, 3, and 5 days or 1 day after focal cerebral ischemia in mice. Motor coordination deficits, brain injury, immune responses in peripheral blood and brain, and cerebral angiogenesis and neurogenesis were analyzed. Postischemic immunosuppression was attenuated in peripheral blood 6 days after ischemia, providing an appropriate external milieu for successful brain remodeling. Although the initial concepts of stem cell therapy aimed at replacing lost tissue, more recent evidence has suggested that stem and progenitor cells alike promote postischemic neurological recovery by secreted factors that restore the injured brain's capacity to reshape. Specifically, extracellular vesicles (EVs) derived from stem cells such as exosomes have recently been suggested to mediate restorative stem cell effects. In order to define whether EVs indeed improve postischemic neurological impairment and brain remodeling, we systematically compared the effects of mesenchymal stem cell (MSC)‐derived EVs (MSC‐EVs) with MSCs that were i.v. delivered to mice on days 1, 3, and 5 (MSC‐EVs) or on day 1 (MSCs) after focal cerebral ischemia in C57BL6 mice. For as long as 28 days after stroke, motor coordination deficits, histological brain injury, immune responses in the peripheral blood and brain, and cerebral angiogenesis and neurogenesis were analyzed. Improved neurological impairment and long‐term neuroprotection associated with enhanced angioneurogenesis were noticed in stroke mice receiving EVs from two different bone marrow‐derived MSC lineages. MSC‐EV administration closely resembled responses to MSCs and persisted throughout the observation period. Although cerebral immune cell infiltration was not affected by MSC‐EVs, postischemic immunosuppression (i.e., B‐cell, natural killer cell, and T‐cell lymphopenia) was attenuated in the peripheral blood at 6 days after ischemia, providing an appropriate external milieu for successful brain remodeling. Because MSC‐EVs have recently been shown to be apparently safe in humans, the present study provides clinically relevant evidence warranting rapid proof‐of‐concept studies in stroke patients. Significance Transplantation of mesenchymal stem cells (MSCs) offers an interesting adjuvant approach next to thrombolysis for treatment of ischemic stroke. However, MSCs are not integrated into residing neural networks but act indirectly, inducing neuroprotection and promoting neuroregeneration. Although the mechanisms by which MSCs act are still elusive, recent evidence has suggested that extracellular vesicles (EVs) might be responsible for MSC‐induced effects under physiological and pathological conditions. The present study has demonstrated that EVs are not inferior to MSCs in a rodent stroke model. EVs induce long‐term neuroprotection, promote neuroregeneration and neurological recovery, and modulate peripheral post‐stroke immune responses. Also, because EVs are well‐tolerated in humans, as previously reported, the administration of EVs under clinical settings might set the path for a novel and innovative therapeutic stroke concept without the putative side effects attached to stem cell transplantation.

The stability of in vitro cell cultures is an important issue for any clinical, bio‐industrial, or pharmacological use. Embryonic stem cells are pluripotent; consequently, they possess the ability to differentiate into all three germ layers and are inherently prone to respond to differentiation stimuli. However, long‐term culture inevitably yields clones that are best adapted to the culture conditions, passaging regimes, or differentiation sensitivity. This cellular plasticity is a major obstacle in the development of bio‐industrial or clinical‐grade cultures. At present, the quality control of cell cultures is limited by the lack of reliable (epi)genetic or molecular markers or by the focus on a particular type of instability such as karyotype abnormalities or adverse phenotypic traits. Therefore, there is an ongoing need for robust, feasible, and sensitive methods of determining or confirming cell status and for revealing potential divergences from the optimal state. We modeled both intrinsic and extrinsic changes in human embryonic stem cell (hESC) states using different experimental strategies and addressed the changes in cell status by intact cell mass spectrometry fingerprinting. The analysis of spectral fingerprints by methods routinely used in analytical chemistry clearly distinguished the morphologically and biochemically similar populations of hESCs and provided a biomarker‐independent tool for the quality control of cell culture. Stem Cells Translational Medicine 2018;7:109–114 Unraveling of hidden alterations to cell phenotype by intact cell mass spectrometry and artificial neural networks as a quality control tool with biomedical applicability – workflow 1 to 4.

As a powerful regulator of cellular homeostasis and metabolism, adenosine is involved in diverse neurological processes including pain, cognition, and memory. Altered adenosine homeostasis has also been associated with several diseases such as depression, schizophrenia, or epilepsy. Based on its protective properties, adenosine has been considered as a potential therapeutic agent for various brain disorders. Since systemic application of adenosine is hampered by serious side effects such as vasodilatation and cardiac suppression, recent studies aim at improving local delivery by depots, pumps, or cell‐based applications. Here, we report on the characterization of adenosine‐releasing human embryonic stem cell‐derived neuroepithelial stem cells (long‐term self‐renewing neuroepithelial stem [lt‐NES] cells) generated by zinc finger nuclease (ZFN)‐mediated knockout of the adenosine kinase (ADK) gene. ADK‐deficient lt‐NES cells and their differentiated neuronal and astroglial progeny exhibit substantially elevated release of adenosine compared to control cells. Importantly, extensive adenosine release could be triggered by excitation of differentiated neuronal cultures, suggesting a potential activity‐dependent regulation of adenosine supply. Thus, ZFN‐modified neural stem cells might serve as a useful vehicle for the activity‐dependent local therapeutic delivery of adenosine into the central nervous system. Stem Cells Translational Medicine 2018;7:477–486 Site‐specific disruption of adenosine kinase results in the augmented release of adenosine into the extracellular space. The amount of secreted adenosine increases during differentiation of neuroepithelial stem cells into neurons and is even further enhanced upon excitatory stimulation. Such an engineered cell population could release neurosuppressive adenosine on demand when high neuronal activity is present, for example in epilepsy.

Abstract Intrauterine adhesions (IUA), which is characterized by endometrial fibrosis, continue to be the most common cause of uterine infertility globally. Our work revealed that 3 fibrotic progression markers (Vimentin, COL5A2, and COL1A1) were significantly increased in the endometrium of IUA patients. Mesenchymal stem cell–derived exosomes (EXOs) have been recently revealed as a cell-free therapy for fibrosis diseases. Nevertheless, the application of EXOs is restricted by the short residency duration in the target tissue. To overcome this limitation, herein, we reported an exosome–based regimen (EXOs-HP) that thermosensitive poloxamer hydrogel possessed the ability to efficiently promote the residency duration of EXOs in the uterine cavity. By downregulating fibrotic progression markers (Vimentin, COL5A2, and COL1A1), EXOs-HP could significantly restore the function and structure of the injured endometrium in the IUA model. Our work provides the theoretical and experimental foundation of EXOs-HP in treating IUA, highlighting the clinical potential of topical EXOs-HP delivery system in IUA patients. Graphical Abstract In this study, we identified 3 upregulated fibrotic progression markers (Vimentin, COL5A2, and COL1A1) in the endometrium of IUA patients and highlighted the efficacious therapy of EXOs–HP via inhibiting the fibrosis process.

Mesenchymal stem cell (MSC)-based therapies are under broad investigation for applications in tissue repair but suffer from poor cell persistence and engraftment upon transplantation. MSC spheroids exhibit improved survival, anti-inflammatory, and angiogenic potential in vitro, while also promoting vascularization when implanted in vivo. However, these benefits are lost once cells engage the tissue extracellular matrix and migrate from the aggregate. The efficacy of cell therapy is consistently improved when using engineered materials, motivating the need to investigate the role of biomaterials to instruct spheroid function. In order to assess the contribution of adhesivity on spheroid activity in engineered materials and promote the bone-forming potential of MSCs, we compared the function of MSC spheroids when entrapped in Arg-Gly-Asp (RGD)-modified alginate hydrogels to nonfouling unmodified alginate. Regardless of material, MSC spheroids exhibited reduced caspase activity and greater vascular endothelial growth factor (VEGF) secretion compared with equal numbers of dissociated cells. MSC spheroids in RGD-modified hydrogels demonstrated significantly greater cell survival than spheroids in unmodified alginate. After 5 days in culture, spheroids in RGD-modified gels had similar levels of apoptosis, but more than a twofold increase in VEGF secretion compared with spheroids in unmodified gels. All gels contained mineralized tissue 8 weeks after subcutaneous implantation, and cells entrapped in RGD-modified alginate exhibited greater mineralization versus cells in unmodified gels. Immunohistochemistry confirmed more diffuse osteocalcin staining in gels containing spheroids compared with dissociated controls. This study demonstrates the promise of cell-instructive biomaterials to direct survival and function of MSC spheroids for bone tissue engineering applications. Mesenchymal stem cell (MSC) spheroids exhibit improved therapeutic potential in vitro compared with dissociated MSCs, yet spheroids are directly injected into tissues, ceding control of cell function to the extracellular matrix and potentially limiting the duration of improvement. Cell delivery using adhesive biomaterials promotes cell retention and function. These studies explored the role of adhesion to the surrounding matrix on spheroid function. When entrapped in an adhesive biomaterial, MSC spheroids exhibited improved survival and proangiogenic growth factor secretion in vitro and bone formation in vivo compared with cells in nonadhesive hydrogels. These findings demonstrate the value of deploying MSC spheroids in instructive biomaterials to improve cell function.

Spinal cord injuries (SCIs) result in devastating lifelong disability for patients and their families. The initial mechanical trauma is followed by a damaging secondary injury cascade involving proapoptotic signaling, ischemia, and inflammatory cell infiltration. Ongoing cellular necrosis releases ATP, DNA, glutamate, and free radicals to create a cytotoxic postinjury milieu. Long‐term regeneration of lost or injured networks is further impeded by cystic cavitation and the formation of an inhibitory glial‐chondroitin sulfate proteoglycan scar. In this article, we discuss important neuroprotective interventions currently applied in clinical practice, including surgical decompression, blood pressure augmentation, and i.v. methylprednisolone. We then explore exciting translational therapies on the horizon, such as riluzole, minocycline, fibroblast growth factor, magnesium, and hypothermia. Finally, we summarize the key neuroregenerative strategies of the next decade, including glial scar degradation, Rho‐ROCK inhibition, cell‐based therapies, and novel bioengineered adjuncts. Throughout, we emphasize the need for combinatorial approaches to this multifactorial problem and discuss relevant studies at the forefront of translation. We conclude by providing our perspectives on the future direction of SCI research. Significance Spinal cord injuries (SCIs) result in devastating, lifelong disability for patients and their families. This article discusses important neuroprotective interventions currently applied in clinical practice, including surgical decompression, blood pressure augmentation, and i.v. methylprednisolone. Translational therapies on the horizon are discussed, such as riluzole, minocycline, fibroblast growth factor, magnesium, and hypothermia. The key neuroregenerative strategies of the next decade are summarized, including glial scar degradation, Rho‐ROCK inhibition, cell‐based therapies, and novel bioengineered adjuncts. The need for combinatorial approaches to this multifactorial problem is emphasized, relevant studies at the forefront of translation are discussed, and perspectives on the future direction of SCI research are presented. This review discusses neuroprotective interventions currently applied in clinical practice for the treatment of spinal cord injuries, as well as translational therapies and neuroregenerative strategies in development. The need for combinatorial approaches to this multifactorial problem is emphasized.