Shi-Jiang Lu's research while affiliated with Ocata Therapeutics and other places

Aim: To investigate the role of DNA methylation during erythrocyte production by human embryonic stem cells (hESCs). Methods: We employed an erythroid differentiation model from hESCs, and then tracked the genome-wide DNA methylation maps and gene expression patterns through an Infinium HumanMethylation450K BeadChip and an Ilumina Human HT-12 v4 Expression Beadchip, respectively. Results: A negative correlation between DNA methylation and gene expression was substantially enriched during the later differentiation stage and was present in both the promoter and the gene body. Moreover, erythropoietic genes with differentially methylated CpG sites that were primarily enriched in nonisland regions were upregulated, and demethylation of their gene bodies was associated with the presence of enhancers and DNase I hypersensitive sites. Finally, the components of JAK-STAT-NF-κB signaling were DNA hypomethylated and upregulated, which targets the key genes for erythropoiesis. Conclusion: Erythroid lineage commitment by hESCs requires genome-wide DNA methylation modifications to remodel gene expression dynamics.

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.

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.

Hemangioblasts are progenitors with the capacity to differentiate into hematopoietic, endothelial, smooth muscle, and mesenchymal stromal cells and represent an excellent candidate of cell therapy for a variety of human diseases. To realize their clinical potential, first, an efficient and controlled differentiation toward hemangioblasts in a scalable manner, probably in a bioreactor setting, from an unlimited source is required. These cells also need to be generated under animal components and cell-free conditions, or additional regulatory challenges, such as xeno-transplant, will have to be addressed. The ability of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSC) to divide indefinitely without losing pluripotency may allow them to serve as an inexhaustible source for the large-scale production of therapeutic cells. In this chapter, we describe a robust system that can efficiently generate large numbers of hemangioblasts from multiple hESCs and iPSC lines under well-defined conditions, which is an important step for future clinical applications, with the potential of developing a GMP-compatible scalable system.

Shortage of red blood cells (RBCs, erythrocytes) can have potentially life-threatening consequences for rare or unusual blood type patients with massive blood loss resulting from various conditions. Erythrocytes have been derived from human pluripotent stem cells (PSCs), but the risk of potential tumorigenicity cannot be ignored, and a majority of these cells produced from PSCs express embryonic ε- and fetal γ-globins with little or no adult β-globin and remain nucleated. Here we report a method to generate erythrocytes from human hair follicle mesenchymal stem cells (hHFMSCs) by enforcing OCT4 gene expression and cytokine stimulation. Cells generated from hHFMSCs expressed mainly the adult β-globin chain with minimum level of the fetal γ-globin chain. Furthermore, these cells also underwent multiple maturation events and formed enucleated erythrocytes with a biconcave disc shape. Gene expression analyses showed that OCT4 regulated the expression of genes associated with both pluripotency and erythroid development during hHFMSC transdifferentiation toward erythroid cells. These findings show that mature erythrocytes can be generated from adult somatic cells, which may serve as an alternative source of RBCs for potential autologous transfusion.

Figure S1. OCT4 transduced hHFMSCs give rise to subsets of hematopoietic-like cells. Figure S2. hHFMSC-derived erythroblasts kept healthy development by adding nutritional factors.

Limitations in the supply of transfusable blood components can have potentially life-threatening consequences for patients with massive blood loss, or who suffer from diseases that cause severe anemia. The ability of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to divide indefinitely without losing pluripotency and to theoretically differentiate into any cell type in the body makes them highly attractive cell sources for large-scale regenerative medicine purposes. Many researchers have already been able to differentiate hESCs and/or iPSCs into specific mature blood cell types, including erythroid, myeloid, macrophage, megakaryocyte/platelet and lymphoid cell lineages. For example, hESC-derived red blood cells and platelets are functional in tasks such as oxygen delivery and blood clotting, respectively, and may be able to serve as substitutes for their donor-derived counterparts in emergencies. hESC-derived dendritic cells are functional in antigen-presentation and may be used as off-the-shelf vaccine therapies to stimulate antigen-specific immune responses against cancer cells. The ability to create banks of hESC lines with matched or reduced incompatibility could potentially reduce or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols altogether, for example, (O) RhD− lines for generation of universal red blood cells. Inasmuch as human iPSCs could potentially be produced from a patient’s own cells, all types of blood cells derived from such iPSC lines would be histocompatible with the patient. For transfusion-dependent patients with unusual or rare blood types, particularly those who are alloimmunized, cells generated in this manner could be an invaluable source of transfusable cells. In this chapter, we highlight the biological functions of mature cells of the blood, clinical conditions requiring the transfusion or stimulation of these cells, and the potential for hESC/iPSC derivatives to serve as functional replacements. However, in vitro differentiation systems used to generate these cells will need further optimization before hESC/iPSC-derived blood components can be used clinically.