Human Hemangioblast-Derived Mesenchymal Stem Cells Promote Islet Engraftment in a Minimal Islet Mass Transplantation Model in Mice.

Islet transplantation can restore glycemic control in patients with type 1 diabetes. Using this procedure, the early stages of engraftment are often crucial to long-term islet function, and outcomes are not always successful. Numerous studies have shown that mesenchymal stem cells (MSCs) facilitate islet graft function. However, experimental data can be inconsistent due to variables associated with MSC generation (including donor characteristics and tissue source), thus, demonstrating the need for a well-characterized and uniform cell product before translation to the clinic. Unlike bone marrow- or adipose tissue-derived MSCs, human embryonic stem cell-derived-MSCs (hESC-MSCs) offer an unlimited source of stable and highly-characterized cells that are easily scalable. Here, we studied the effects of human hemangioblast-derived mesenchymal cells (HMCs), (i.e., MSCs differentiated from hESCs using a hemangioblast intermediate), on islet cell transplantation using a minimal islet mass model. The co-transplantation of the HMCs allowed a mass of islets that was insufficient to correct diabetes on its own to restore glycemic control in all recipients. Our in vitro studies help to elucidate the mechanisms including reduction of cytokine stress by which the HMCs support islet graft protection in vivo. Derivation, stability, and scalability of the HMC source may offer unique advantages for clinical applications, including fewer islets needed for successful islet transplantation.

Laser power stabilization via radiation pressure.

This Letter reports the experimental realization of a novel, to the best of our knowledge, active power stabilization scheme in which laser power fluctuations are sensed via the radiation pressure driven motion they induce on a movable mirror. The mirror position and its fluctuations were determined by means of a weak auxiliary laser beam and a Michelson interferometer, which formed the in-loop sensor of the power stabilization feedback control system. This sensing technique exploits a nondemolition measurement, which can result in higher sensitivity for power fluctuations than direct, and hence destructive, detection. Here we used this new scheme in a proof-of-concept experiment to demonstrate power stabilization in the frequency range from 1 Hz to 10 kHz, limited at low frequencies by the thermal noise of the movable mirror at room temperature.

Next-generation stem cells - ushering in a new era of cell-based therapies.

Naturally occurring stem cells isolated from humans have been used therapeutically for decades. This has primarily involved the transplantation of primary cells such as haematopoietic and mesenchymal stem cells and, more recently, derivatives of pluripotent stem cells. However, the advent of cell-engineering approaches is ushering in a new generation of stem cell-based therapies, greatly expanding their therapeutic utility. These next-generation stem cells are being used as 'Trojan horses' to improve the delivery of drugs and oncolytic viruses to intractable tumours and are also being engineered with angiogenic, neurotrophic and anti-inflammatory molecules to accelerate the repair of injured or diseased tissues. Moreover, gene therapy and gene editing technologies are being used to create stem cell derivatives with improved functionality, specificity and responsiveness compared with their natural counterparts. Here, we review these engineering approaches and areas in which they will help broaden the utility and clinical applicability of stem cells.

Engineering universal cells that evade immune detection.

The prospect of transplanting cells and tissues without the risk of immune rejection or the need for powerful immunosuppressive drugs is the 'holy grail' of transplantation medicine. Now, with the advent of pluripotent stem cells, CRISPR-Cas9 and other gene-editing technologies, the race to create 'off-the-shelf' donor cells that are invisible to the immune system ('universal cells') has started. One important approach for creating such cells involves the manipulation of genes required for immune recognition, in particular HLA class I and II proteins. Other approaches leverage knowledge of immune-cloaking strategies used by certain bacteria, viruses, parasites, the fetus and cancer cells to induce tolerance to allogeneic cell-based therapies by modifying cells to express immune-suppressive molecules such as PD-L1 and CTLA4-Ig. Various academic groups as well as biotechnology and pharmaceutical companies are on the verge of bringing these therapies into the clinic.

Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in Macular Degeneration.

PURPOSE: Transplantation of human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells offers the potential for benefit in macular degeneration. Previous trials have reported improved visual acuity (VA), but lacked detailed analysis of retinal structure and function in the treated area. DESIGN: Phase 1/2 open-label dose-escalation trial to evaluate safety and potential efficacy (clinicaltrials.gov identifier, NCT01469832). PARTICIPANTS: Twelve participants with advanced Stargardt disease (STGD1), the most common cause of macular degeneration in children and young adults. METHODS: Subretinal transplantation of up to 200 000 hESC-derived RPE cells with systemic immunosuppressive therapy for 13 weeks. MAIN OUTCOME MEASURES: The primary end points were the safety and tolerability of hESC-derived RPE cell administration. We also investigated evidence of the survival of transplanted cells and measured retinal structure and function using microperimetry and spectral-domain OCT. RESULTS: Focal areas of subretinal hyperpigmentation developed in all participants in a dose-dependent manner in the recipient retina and persisted after withdrawal of systemic immunosuppression. We found no evidence of uncontrolled proliferation or inflammatory responses. Borderline improvements in best-corrected VA in 4 participants either were unsustained or were matched by a similar improvement in the untreated contralateral eye. Microperimetry demonstrated no evidence of benefit at 12 months in the 12 participants. In one instance at the highest dose, localized retinal thinning and reduced sensitivity in the area of hyperpigmentation suggested the potential for harm. Participant-reported quality of life using the 25-item National Eye Institute Visual Function Questionnaire indicated no significant change. CONCLUSIONS: Subretinal hyperpigmentation is consistent with the survival of viable transplanted hESC-derived RPE cells, but may reflect released pigment in their absence. The findings demonstrate the value of detailed analysis of spatial correlation of retinal structure and function in determining with appropriate sensitivity the impact of cell transplantation and suggest that intervention in early stage of disease should be approached with caution. Given the slow rate of progressive degeneration at this advanced stage of disease, any protection against further deterioration may be evident only after a more extended period of observation.

Human Embryonic Stem Cell-Derived Mesenchymal Stromal Cells Decrease the Development of Severe Experimental Autoimmune Uveitis in B10.RIII Mice.

PURPOSE: We investigated the effect of exogenously administered human embryonic stem cell-derived mesenchymal stromal cells (hESC-MSCs) in experimental autoimmune uveitis (EAU) in B10.RIII mice, a murine model of severe uveitis. METHODS: B10.RIII mice were immunized with an uveitogenic peptide, and intraperitoneal injections of 5 million hESC-MSCs per animal were given on the same day. Behavioral light sensitivity assays, histological evaluation, cytokine production, and regulatory T cells were analyzed at the peak of the disease. RESULTS: Histological and behavioral evidence demonstrated that early systemic treatment with hESC-MSCs decreases the development of severe EAU in B10.RIII mice. hESC-MSCs suppress Th17 and upregulate Th1 and Th2 responses as well as IL-2 and GM-CSF in splenocytes from hESC-MSC-treated mice. CONCLUSIONS: MSCs that originate from hESC decrease the development of severe EAU in B10.RIII mice, likely through systemic immune modulation. Further investigation is needed to determine any potential effect on active EAU.

Retinal vascular injuries and intravitreal human embryonic stem cell-derived haemangioblasts.

OBJECTIVE: To investigate whether intravitreally applied haemangioblasts (HB) derived from human embryonic stem cells (hESCs) are helpful for the repair of vascular damage caused in animals by an oxygen-induced retinopathy (OIR), by an induced diabetic retinopathy (DR) or by an induced retinal ischaemia with subsequent reperfusion. METHODS: Human embryonic stem cell-derived HBs were transplanted intravitreally into C57BL/6J mice (OIR model), into male Wistar rats with an induced DR and into male Wistar rats undergoing induced retinal ischaemia with subsequent reperfusion. Control groups of animals received an intravitreal injection of endothelial cells (ECs) or phosphate-buffered saline (PBS). We examined the vasculature integrity in the mice with OIR, the blood-retina barrier in the rats with induced DR, and retinal thickness and retinal ganglion cell density in retina flat mounts of the rats with the retinal ischaemic-reperfusion retinopathy. RESULTS: In the OIR model, the study group versus control groups showed a significantly (p < 0.001) smaller retinal avascular area [5.1 ± 2.7%;n = 18 animals versus 12.2 ± 2.8% (PBS group; n = 10 animals) and versus 11.8 ± 3.7% (EC group; n = 8 animals)] and less retinal neovascularization [6.3 ± 2.5%;n = 18 versus 15.2 ± 6.3% (n = 10; PBS group) and versus 15.8 ± 3.3% (n = 8; EC group)]. On retinal flat mounts, hESC-HBs were integrated into damaged retinal vessels and stained positive for PECAM (CD31) as EC marker. In the DR model, the study group versus the EC control group showed a significantly (p = 0.001) better blood-retina barrier function as measured at 2 days after the intravitreal injections [study group: 20.2 ± 12.8 µl/(g × hr); n = 6; versus EC control group: 52.9 ± 9.9 µl/(g × hr; n = 6)]. In the retinal ischaemia-reperfusion model, the groups did not differ significantly in retinal thickness and retinal ganglion cell density at 2, 5 and 7 days after baseline. CONCLUSION: By integrating into damaged retinal vessels and differentiating into ECs, intravitreally administered hESC-HBs may have partially repaired a retinal vascular injury caused by OIR model and DR.

Pluripotent stem cells: the last 10 years.

Pluripotent stem cells (PSCs) can differentiate into virtually any cell type in the body, making them attractive for both regenerative medicine and drug discovery. Over the past 10 years, technological advances and innovative platforms have yielded first-in-man PSC-based clinical trials and opened up new approaches for disease modeling and drug development. Induced PSCs have become the foremost alternative to embryonic stem cells and accelerated the development of disease-in-a-dish models. Over the years and with each new discovery, PSCs have proven to be extremely versatile. This review article highlights key advancements in PSC research, from 2006 to 2016, and how they will guide the direction of the field over the next decade.

Function of human pluripotent stem cell-derived photoreceptor progenitors in blind mice.

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement may hold the potential for repair in a completely degenerate retina by reinstating light sensitive cells to form connections that relay information to downstream retinal layers. This study assessed the therapeutic potential of photoreceptor progenitors derived from human embryonic and induced pluripotent stem cells (ESCs and iPSCs) using a protocol that is suitable for future clinical trials. ESCs and iPSCs were cultured in four specific stages under defined conditions, resulting in generation of a near-homogeneous population of photoreceptor-like progenitors. Following transplantation into mice with end-stage retinal degeneration, these cells differentiated into photoreceptors and formed a cell layer connected with host retinal neurons. Visual function was partially restored in treated animals, as evidenced by two visual behavioral tests. Furthermore, the magnitude of functional improvement was positively correlated with the number of engrafted cells. Similar efficacy was observed using either ESCs or iPSCs as source material. These data validate the potential of human pluripotent stem cells for photoreceptor replacement therapies aimed at photoreceptor regeneration in retinal disease.

Treatment of perianal fistulas with human embryonic stem cell-derived mesenchymal stem cells: a canine model of human fistulizing Crohn's disease.

AIM: To evaluate the safety and efficacy of intralesional injection of human embryonic stem cell (hESC)-derived mesenchymal stem/stromal cells (MSCs) in canine anal furunculosis dogs. MATERIALS & METHODS: Dogs naturally develop an immune-mediated disease called canine anal furunculosis, which shares many features with human fistulizing Crohn's disease. RESULTS: The hESC-MSCs were well tolerated and 1 month postinjection, accompanied by reduced serum levels of IL-2 and IL-6, two inflammatory cytokines associated with Crohn's disease. All six dogs were found to be completely free of fistulas at 3 months postinjection. However, at 6 months, two dogs had some fistula relapse. CONCLUSION: Results of this study provide the first evidence of the safety and therapeutic potential of hESC-MSCs in a large animal model.

Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.

AIM: To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. MATERIALS AND METHODS: Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression. RESULTS: hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPaseα1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis. CONCLUSION: hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

Human embryonic stem cell-derived mesenchymal cells preserve kidney function and extend lifespan in NZB/W F1 mouse model of lupus nephritis.

Adult tissue-derived mesenchymal stromal cells (MSCs) are showing promise in clinical trials for systemic lupus erythematosus (SLE). However, the inability to manufacture large quantities of functional cells from a single donor as well as donor-dependent variability in quality limits their clinical utility. Human embryonic stem cell (hESC)-derived MSCs are an alternative to adult MSCs that can circumvent issues regarding scalability and consistent quality due to their derivation from a renewable starting material. Here, we show that hESC-MSCs prevent the progression of fatal lupus nephritis (LN) in NZB/W F1 (BWF1) mice. Treatment led to statistically significant reductions in proteinuria and serum creatinine and preserved renal architecture. Specifically, hESC-MSC treatment prevented disease-associated interstitial inflammation, protein cast deposition, and infiltration of CD3(+) lymphocytes in the kidneys. This therapy also led to significant reductions in serum levels of tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6), two inflammatory cytokines associated with SLE. Mechanistically, in vitro data support these findings, as co-culture of hESC-MSCs with lipopolysaccharide (LPS)-stimulated BWF1 lymphocytes decreased lymphocyte secretion of TNFα and IL-6, and enhanced the percentage of putative regulatory T cells. This study represents an important step in the development of a commercially scalable and efficacious cell therapy for SLE/LN.

Current status of pluripotent stem cells: moving the first therapies to the clinic.

Pluripotent stem cells (PSCs) hold great promise for drug discovery and regenerative medicine owing to their ability to differentiate into any cell type in the body. After more than three decades of research, including delays due to the potential tumorigenicity of PSCs and inefficiencies in differentiation methods, the field is at a turning point, with a number of clinical trials across the globe now testing PSC-derived products in humans. Ocular diseases dominate these first-in-man trials, and Phase l/ll results are showing promising safety data as well as possible efficacy. In addition, the advent of induced PSC (iPSC) technology is enabling the development of a wide range of cell-based disease models from genetically predisposed patients, thereby facilitating drug discovery. In this Review, we discuss the recent progress and remaining challenges for the use of PSCs in regenerative medicine and drug development.

Treatment of macular degeneration using embryonic stem cell-derived retinal pigment epithelium: preliminary results in Asian patients.

Embryonic stem cells hold great promise for various diseases because of their unlimited capacity for self-renewal and ability to differentiate into any cell type in the body. However, despite over 3 decades of research, there have been no reports on the safety and potential efficacy of pluripotent stem cell progeny in Asian patients with any disease. Here, we report the safety and tolerability of subretinal transplantation of human embryonic-stem-cell (hESC)-derived retinal pigment epithelium in four Asian patients: two with dry age-related macular degeneration and two with Stargardt macular dystrophy. They were followed for 1 year. There was no evidence of adverse proliferation, tumorigenicity, ectopic tissue formation, or other serious safety issues related to the transplanted cells. Visual acuity improved 9-19 letters in three patients and remained stable (+1 letter) in one patient. The results confirmed that hESC-derived cells could serve as a potentially safe new source for regenerative medicine.

Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt's macular dystrophy: follow-up of two open-label phase 1/2 studies.

BACKGROUND: Since they were first derived more than three decades ago, embryonic stem cells have been proposed as a source of replacement cells in regenerative medicine, but their plasticity and unlimited capacity for self-renewal raises concerns about their safety, including tumour formation ability, potential immune rejection, and the risk of differentiating into unwanted cell types. We report the medium-term to long-term safety of cells derived from human embryonic stem cells (hESC) transplanted into patients. METHODS: In the USA, two prospective phase 1/2 studies were done to assess the primary endpoints safety and tolerability of subretinal transplantation of hESC-derived retinal pigment epithelium in nine patients with Stargardt's macular dystrophy (age >18 years) and nine with atrophic age-related macular degeneration (age >55 years). Three dose cohorts (50,000, 100,000, and 150,000 cells) were treated for each eye disorder. Transplanted patients were followed up for a median of 22 months by use of serial systemic, ophthalmic, and imaging examinations. The studies are registered with ClinicalTrials.gov, numbers NCT01345006 (Stargardt's macular dystrophy) and NCT01344993 (age-related macular degeneration). FINDINGS: There was no evidence of adverse proliferation, rejection, or serious ocular or systemic safety issues related to the transplanted tissue. Adverse events were associated with vitreoretinal surgery and immunosuppression. 13 (72%) of 18 patients had patches of increasing subretinal pigmentation consistent with transplanted retinal pigment epithelium. Best-corrected visual acuity, monitored as part of the safety protocol, improved in ten eyes, improved or remained the same in seven eyes, and decreased by more than ten letters in one eye, whereas the untreated fellow eyes did not show similar improvements in visual acuity. Vision-related quality-of-life measures increased for general and peripheral vision, and near and distance activities, improving by 16-25 points 3-12 months after transplantation in patients with atrophic age-related macular degeneration and 8-20 points in patients with Stargardt's macular dystrophy. INTERPRETATION: The results of this study provide the first evidence of the medium-term to long-term safety, graft survival, and possible biological activity of pluripotent stem cell progeny in individuals with any disease. Our results suggest that hESC-derived cells could provide a potentially safe new source of cells for the treatment of various unmet medical disorders requiring tissue repair or replacement. FUNDING: Advanced Cell Technology.

Scalable generation of universal platelets from human induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid "surge" capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the ß2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.

Human ESC-derived MSCs outperform bone marrow MSCs in the treatment of an EAE model of multiple sclerosis.

Current therapies for multiple sclerosis (MS) are largely palliative, not curative. Mesenchymal stem cells (MSCs) harbor regenerative and immunosuppressive functions, indicating a potential therapy for MS, yet the variability and low potency of MSCs from adult sources hinder their therapeutic potential. MSCs derived from human embryonic stem cells (hES-MSCs) may be better suited for clinical treatment of MS because of their unlimited and stable supply. Here, we show that hES-MSCs significantly reduce clinical symptoms and prevent neuronal demyelination in a mouse experimental autoimmune encephalitis (EAE) model of MS, and that the EAE disease-modifying effect of hES-MSCs is significantly greater than that of human bone-marrow-derived MSCs (BM-MSCs). Our evidence also suggests that increased IL-6 expression by BM-MSCs contributes to the reduced anti-EAE therapeutic activity of these cells. A distinct ability to extravasate and migrate into inflamed CNS tissues may also be associated with the robust therapeutic effects of hES-MSCs on EAE.

Human somatic cell nuclear transfer using adult cells.

Derivation of patient-specific human pluripotent stem cells via somatic cell nuclear transfer (SCNT) has the potential for applications in a range of therapeutic contexts. However, successful SCNT with human cells has proved challenging to achieve, and thus far has only been reported with fetal or infant somatic cells. In this study, we describe the application of a recently developed methodology for the generation of human ESCs via SCNT using dermal fibroblasts from 35- and 75-year-old males. Our study therefore demonstrates the applicability of SCNT for adult human cells and supports further investigation of SCNT as a strategy for regenerative medicine.