Ten years of iPSC: clinical potential and advances in vitro hematopoietic differentiation

Ten years have passed since the first publication announcing the generation of induced pluripotent stem cells (iPSCs). Issues related to ethics, immune rejection, and cell availability seemed to be solved following this breakthrough. The development of iPSC technology allows advances in in vitro cell differentiation for cell therapy purpose and other clinical applications. This review provides a perspective on the iPSC potential for cell therapies, particularly for hematological applications. We discuss the advances in in vitro hematopoietic differentiation, the possibilities to employ iPSC in hematology studies, and their potential clinical application in hematologic diseases. The generation of red blood cells and functional T cells and the genome editing technology applied to mutation correction are also covered. We highlight some of the requirements and obstacles to be overcome before translating these cells from research to the clinic, for instance, iPSC variability, genotoxicity, the differentiation process, and engraftment. Also, we evaluate the patent landscape and compile the clinical trials in the field of pluripotent stem cells. Currently, we know much more about iPSC than in 2006, but there are still challenges that must be solved. A greater understanding of molecular mechanisms underlying the generation of hematopoietic stem cells is necessary to produce suitable and transplantable hematopoietic stem progenitor cells from iPSC.     

A Versatile Tool for the Quantification of CRISPR/Cas9-Induced Genome Editing Events in Human Hematopoietic Cell Lines and Hematopoietic Stem/Progenitor Cells

The efficient site-specific DNA double-strand breaks (DSB) created by CRISPR/Cas9 has revolutionized genome engineering and has great potential for editing hematopoietic stem/progenitor cells (HSPCs). However, detailed understanding of the variables that influence choice of DNA–DSB repair (DDR) pathways by HSPC is required for therapeutic levels of editing in these clinically relevant cells. We developed a hematopoietic-reporter system that rapidly quantifies the three major DDR pathways utilized at the individual DSB created by CRISPR/Cas9—NHEJ, MMEJ, and HDR—and show its applicability in evaluating the different DDR outcomes utilized by human hematopoietic cell lines and primary human HSPC.     

CRISPR/Cas9-mediated gene editing. A promising strategy in hematological disorders

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the gene editing field, making it possible to interrupt, insert or replace a sequence of interest with high precision in the human genome. Its easy design and wide applicability open up a variety of therapeutic alternatives for the treatment of genetic diseases. Indeed, very promising approaches for the correction of hematological disorders have been developed in the recent years, based on the self-renewal and multipotent differentiation properties of hematopoietic stem and progenitor cells, which make this cell subset the ideal target for gene therapy purposes. This technology has been applied in different congenital blood disorders, such as primary immunodeficiencies, X-linked severe combined immunodeficiency, X-linked chronic granulomatous disease or Wiskott–Aldrich syndrome, and inherited bone marrow failure syndromes, such as Fanconi anemia, congenital amegakaryocytic thrombocytopenia or severe congenital neutropenia. Furthermore, CRISPR/Cas9-based gene editing has been implemented successfully as a novel therapy for cancer immunotherapy, by the development of promising strategies such as the use of oncolytic viruses or adoptive cellular therapy to the chimeric antigen receptor–T-cell therapy. Therefore, considering the variety of genes and mutations affected, we can take advantage of the different DNA repair mechanisms by CRISPR/Cas9 in different manners, from homology-directed repair to non-homologous-end-joining to the latest emerging technologies such as base and prime editing. Although the delivery systems into hematopoietic stem and progenitor cells are still the bottleneck of this technology, some of the advances in genome editing shown in this review have already reached a clinical stage and show very promising preliminary results.     

Convergence of human pluripotent stem cell,organoid, and genome editing technologies

The last decade has seen many exciting technological breakthroughs that greatly expanded the toolboxes for biological and biomedical research, yet few have had more impact than induced pluripotent stem cells and modern-day genome editing. These technologies are providing unprecedented opportunities to improve physiological relevance of experimental models, further our understanding of developmental processes, and develop novel therapies. One of the research areas that benefit greatly from these technological advances is the three-dimensional human organoid culture systems that resemble human tissues morphologically and physiologically. Here we summarize the development of human pluripotent stem cells and their differentiation through organoid formation. We further discuss how genetic modifications, genome editing in particular, were applied to answer basic biological and biomedical questions using organoid cultures of both somatic and pluripotent stem cell origins. Finally, we discuss the potential challenges of applying human pluripotent stem cell and organoid technologies for safety and efficiency evaluation of emerging genome editing tools.     

Stabilins are expressed in bone marrow sinusoidal endothelial cells and mediate scavenging and cell adhesive functions

Bone marrow sinusoidal endothelial cells have a specific function as a site of transmigration of hematopoietic stem and progenitor cells and mature blood cells between bone marrow and blood stream. However, the specific characteristics of bone marrow sinusoidal endothelial cells are still largely unclear. We here report that these cells express stabilin-1 and stabilin-2, which in liver sinusoidal endothelial cells have been identified as endocytic scavenger receptors for several ligands, including SPARC and hyaluronan. We show here that intravenously injected formaldehyde-treated serum albumin, advanced glycation end-products, and collagen I α-chains were taken up by bone marrow sinusoidal endothelial cells, showing that these cells have a scavenging function and thereby may modulate bone marrow vascular stem cell niches. Importantly, we show hyaluronan mediated adhesion of hematopoietic stem and progenitor cells to stabilin-2-transfected cells, suggesting that stabilin-2 contributes to adhesion and homing of circulating stem and progenitor cells to bone marrow.     

β-血红蛋白病基因组编辑治疗的研究进展

β-血红蛋白病(β-hemoglobinopathies)是严重危害人类健康的6种常见疾病之一,尽管其遗传分子机制已被阐释清楚,但目前除异基因骨髓移植外尚还缺乏根治性的治疗策略。近年来,基因组编辑技术的迅速发展及其在人造血干祖细胞中的应用为β-血红蛋白病的治疗开辟了新的方向。针对血红蛋白的基因突变,可利用同源重组介导的细胞内源性DNA损伤修复途径直接修复遗传缺陷,也可以利用非同源末端连接机制沉默抑制胎儿血红蛋白表达的分子,重新激活胎儿血红蛋白的表达以缓解β-血红蛋白病人的临床症状。本文从β-血红蛋白病的基因组编辑策略及尝试、临床转化平台的要素分析等方面对该病的基因组编辑治疗的研究进展展开综述,以期为β-血红蛋白病新型治愈方案的研究以及基因组编辑技术的临床转化提供参考。     

Promotion of hematopoietic differentiation from mouse induced pluripotent stem cells by transient HoxB4 transduction

Ectopic expression of HoxB4 in embryonic stem (ES) cells leads to an efficient production of hematopoietic cells, including hematopoietic stem/progenitor cells. Previous studies have utilized a constitutive HoxB4 expression system or tetracycline-regulated HoxB4 expression system to induce hematopoietic cells from ES cells. However, these methods cannot be applied therapeutically due to the risk of transgenes being integrated into the host genome. Here, we report the promotion of hematopoietic differentiation from mouse ES cells and induced pluripotent stem (iPS) cells by transient HoxB4 expression using an adenovirus (Ad) vector. Ad vector could mediate efficient HoxB4 expression in ES cell-derived embryoid bodies (ES-EBs) and iPS-EBs, and its expression was decreased during cultivation, showing that Ad vector transduction was transient. A colony-forming assay revealed that the number of hematopoietic progenitor cells with colony-forming potential in HoxB4-transduced cells was significantly increased in comparison with that in non-transduced cells or LacZ-transduced cells. HoxB4-transduced cells also showed more efficient generation of CD41-, CD45-, or Sca-1-positive cells than control cells. These results indicate that transient, but not constitutive, HoxB4 expression is sufficient to augment the hematopoietic differentiation of ES and iPS cells, and that our method would be useful for clinical applications, such as cell transplantation therapy.     

Umbilical cord blood stem cells: Towards a proteomic approach

The first umbilical cord blood (UCB) transplant to a sibling with Fanconi's anaemia in 1988 represented a breakthrough in the field of transplantation. Thereon, several transplants have been performed with UCB-derived hematopoietic stem cells (HSCs) and a plethora of studies have investigated the plasticity of UCB-derived stem and progenitor cells. However, these studies have not been hitherto translated into clinical trials and, although UCB is routinely used as an alternative source of HSCs, no substantial advances have been made in the field of clinical regenerative medicine. The real deal is the lack of knowledge about the molecular processes governing the events of differentiation which transform immature UCB stem cells into terminally-committed hematopoietic, muscle, bone and nervous cells. In order to fill this void, several studies have been recently focused on the identification of the peculiar proteomic profile of UCB-derived stem cells.Hereby, we concisely review recent proteomic surveys addressing UCB-derived stem and progenitor cells.Notably, comparative studies detected a wider spectrum of proteins in immature cells rather than in more differentiated populations, as if maturation events could represent a bottleneck to protein expression. Future research projects should try to shed light on these processes and their completion could pave the way for unprecedented treatments.     

Large-scale genome editing based on high-capacity adenovectors and CRISPR-Cas9 nucleases rescues full-length dystrophin synthesis in DMD muscle cells

Targeted chromosomal insertion of large genetic payloads in human cells leverages and broadens synthetic biology and genetic therapy efforts. Yet, obtaining large-scale gene knock-ins remains particularly challenging especially in hard-to-transfect stem and progenitor cells. Here, fully viral gene-deleted adenovector particles (AdVPs) are investigated as sources of optimized high-specificity CRISPR-Cas9 nucleases and donor DNA constructs tailored for targeted insertion of full-length dystrophin expression units (up to 14.8-kb) through homologous recombination (HR) or homology-mediated end joining (HMEJ). In muscle progenitor cells, donors prone to HMEJ yielded higher CRISPR-Cas9-dependent genome editing frequencies than HR donors, with values ranging between 6% and 34%. In contrast, AdVP transduction of HR and HMEJ substrates in induced pluripotent stem cells (iPSCs) resulted in similar CRISPR-Cas9-dependent genome editing levels. Notably, when compared to regular iPSCs, in p53 knockdown iPSCs, CRISPR-Cas9-dependent genome editing frequencies increased up to 6.7-fold specifically when transducing HMEJ donor constructs. Finally, single DNA molecule analysis by molecular combing confirmed that AdVP-based genome editing achieves long-term complementation of DMD-causing mutations through the site-specific insertion of full-length dystrophin expression units. In conclusion, AdVPs are a robust and flexible platform for installing large genomic edits in human cells and p53 inhibition fosters HMEJ-based genome editing in iPSCs.     

Keratinocyte stem cells of cornea,skin and hair follicle: common and distinguishing features

Tissues which undergo constant regeneration have developed a complex and tightly regulated system of replication and differentiation. The most primitive progenitor cells in these populations are termed stem cells. Much has been learned in recent years about the nature of stem cells and the dynamics of their replication, especially within hematopoietic tissues. This paper will define eight general features of stem cells, as they have been characterized in hematopoietic populations, and then review their presence in several keratinocyte populations, including corneal epithelium, interfollicular epidermis and hair follicle. Finally, this review will raise several important questions that remain unanswered about the nature of keratinocyte stem cells and hopefully will be the focus of future research.     

Pleiotropic effects of prostaglandin E2 in hematopoiesis; prostaglandin E2 and other eicosanoids regulate hematopoietic stem and progenitor cell function

Eicosanoids have been implicated in the physiological regulation of hematopoiesis with pleiotropic effects on hematopoietic stem cells and various classes of lineage restricted progenitor cells. Herein we review the effects of eicosanoids on hematopoiesis, focusing on new findings implicating prostaglandin E(2) in enhancing hematopoietic stem cell engraftment by enhancing stem cell homing, survival and self-renewal. We also describe a role for cannabinoids in hematopoiesis. Lastly, we discuss the yin and yang of various eicosanoids in modulating hematopoietic stem and progenitor cell functions and summarize potential strategies to take advantage of these effects for therapeutic benefit for hematopoietic stem cell transplantation.     

Placental Growth Factor Expression Is Required for Bone Marrow Endothelial Cell Support of Primitive Murine Hematopoietic Cells

Two distinct microenvironmental niches that regulate hematopoietic stem/progenitor cell physiology in the adult bone marrow have been proposed; the endosteal and the vascular niche. While extensive studies have been performed relating to molecular interactions in the endosteal niche, the mechanisms that regulate hematopoietic stem/progenitor cell interaction with bone marrow endothelial cells are less well defined. Here we demonstrate that endothelial cells derived from the bone marrow supported hematopoietic stem/progenitor cells to a higher degree than other endothelial or stromal cell populations. This support was dependant upon placental growth factor expression, as genetic knockdown of mRNA levels reduced the ability of endothelial cells to support hematopoietic stem/progenitor cells in vitro. Furthermore, using an in vivo model of recovery from radiation induced myelosuppression, we demonstrate that bone marrow endothelial cells were able to augment the recovery of the hematopoietic stem/progenitor cells. However, this effect was diminished when the same cells with reduced placental growth factor expression were administered, possibly owing to a reduced homing of the cells to the bone marrow vasculature. Our data suggest that placental growth factor elaborated from bone marrow endothelial cells mediates the regulatory effects of the vascular niche on hematopoietic stem/progenitor cell physiology.     

A new genetic tool to improve immune‐compromised mouse models: Derivation and CRISPR/Cas9‐mediated targeting of NRG embryonic stem cell lines

Development of human hematopoietic stem cells and differentiation of embryonic stem (ES) cells/induced pluripotent stem (iPS) cells to hematopoietic stem cells are poorly understood. NOD (Non‐obese diabetic)‐derived mouse strains, such as NSG (NOD‐Scid‐il2Rg) or NRG (NOD‐Rag1‐il2Rg), are the best available models for studying the function of fetal and adult human hematopoietic cells as well as ES/iPS cell‐derived hematopoietic stem cells. Unfortunately, engraftment of human hematopoietic stem cells is very variable in these models. Introduction of additional permissive mutations into these complex genetic backgrounds of the NRG/NSG mice by natural breeding is a very demanding task in terms of time and resources. Specifically, since the genetic elements defining the NSG/NRG phenotypes have not yet been fully characterized, intense backcrossing is required to ensure transmission of the full phenotype. Here we describe the derivation of embryonic stem cell (ESC) lines from NRG pre‐implantation embryos generated by in vitro fertilization followed by the CRISPR/CAS9 targeting of the Gata‐2 locus. After injection into morula stage embryos, cells from three tested lines gave rise to chimeric adult mice showing high contribution of the ESCs (70%–100%), assessed by coat color. Moreover, these lines have been successfully targeted using Cas9/CRISPR technology, and the mutant cells have been shown to remain germ line competent. Therefore, these new NRG ESC lines combined with genome editing nucleases bring a powerful genetic tool that facilitates the generation of new NOD‐based mouse models with the aim to improve the existing xenograft models.     

Genome Editing in Mouse Spermatogonial Stem/Progenitor Cells Using Engineered Nucleases

Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease. We used engineered nucleases to stimulate homologous recombination to correct a mutant gene in mouse “GS” (germline stem) cells, testicular derived cell cultures containing spermatogonial stem cells and progenitor cells. We demonstrated that gene-corrected cells maintained several properties of spermatogonial stem/progenitor cells including the ability to colonize following testicular transplantation. This proof of concept for genome editing in GS cells impacts both cell therapy and basic research given the potential for GS cells to be propagated in vitro, contribute to the germline in vivo following testicular transplantation or become reprogrammed to pluripotency in vitro.     

巨核细胞生成调控相关细胞因子及其作用研究进展

造血干细胞分化生成巨核细胞是一个十分复杂的过程,包括造血干细胞动员及其向巨核系祖细胞分化,巨核系祖细胞增殖、分化生成未成熟巨核细胞,巨核细胞的成熟和血小板释放等过程。研究发现,造血干细胞动员及其向各系细胞分化的大部分过程都在一种称为"龛"的结构中进行,多种龛内信号分子参与了造血干细胞的动员和分化调控。该文对造血干细胞龛内参与造血干细胞动员和分化生成巨核细胞的几种重要细胞因子及其调控作用进行综述。     

Hematopoietic growth factors in autologous transplantation

Hematopoietic growth factors (HGFs) sustain the survival, proliferation and differentiation of hematopoietic stem cells and some functions of mature blood cells. In man several HGFs have been characterised and cloned so far, and this has allowed investigators to confer the rationale for the clinical application of these molecules in hematology and oncology. In particular G-CSF and GM-CSF are currently utilised to abrogate the hematological toxicity of chemotherapy for standard and dose-intensified therapy, neutropenia following bone marrow and peripheral blood stem cell transplantation. Moreover there has recently been great interest in the ex vivo expansion of hematopoietic stem and progenitor cells for a variety of applications, such as in vitro tumor cell purging or for reducing the volume of blood processed by the leukapheresis. Several combinations of HGFs have been described to sustain the ex vivo survival and proliferation of these cells disclosing new opportunities in the field of stem cells transplants.     

Mobilization of human hematopoietic stem/progenitor-enriched CD34+ cells into peripheral blood during stress related to ischemic stroke

The bone marrow-derived stem/progenitor cells were demonstrated to play an important role in a regeneration of damaged tissue. Based on these observations we asked whether the stroke-related stress triggers mobilization of stem/progenitor cells from the bone marrow into the peripheral blood, which subsequently could contribute to regeneration of damaged organs. To address this issue, the peripheral blood samples were harvested from patients with ischemic stroke during the first 24 hrs as well as after the 48 (2nd day) and 144 hrs (6th day) since the manifestation of symptoms. In these patients we evaluated the percentage of hematopoietic stem/progenitor-enriched CD34+ cells by employing flow cytometry and the number of hematopoietic progenitor cells for the granulocyto-monocytic (CFU-GM) and erythroid (BFU-E)-lineages circulating in peripheral blood. We concluded that stress related to ischemic stroke triggers the mobilization of hematopoietic stem/progenitor cells from the bone marrow into peripheral blood. These circulating stem/progenitor cells may play an important role in the process of regeneration of the ischemic tissue.