Transcription factor-mediated reprogramming toward hematopoietic stem cells.

De novo generation of human hematopoietic stem cells (HSCs) from renewable cell types has been a long sought-after but elusive goal in regenerative medicine. Paralleling efforts to guide pluripotent stem cell differentiation by manipulating developmental cues, substantial progress has been made recently toward HSC generation via combinatorial transcription factor (TF)-mediated fate conversion, a paradigm established by Yamanaka's induction of pluripotency in somatic cells by mere four TFs. This review will integrate the recently reported strategies to directly convert a variety of starting cell types toward HSCs in the context of hematopoietic transcriptional regulation and discuss how these findings could be further developed toward the ultimate generation of therapeutic human HSCs.

Differentiation of adult human retinal pigment epithelial cells into dopaminergic-like cells in vitro and in the recipient monkey brain.

BACKGROUND: Cell therapy is proposed to be a potential treatment for Parkinson's disease (PD). Although fetal retinal pigment epithelial (RPE) cells have been tested in trials for treating PD patients, controversy has been raised over the issue of whether such cells can be reprogrammed into dopamine-producing cells for therapeutic efficacy. Here, we aim to investigate whether adult human RPE cells can be reprogrammed into dopamine-producing cells both in vitro and in the recipient monkey brain. METHODS: The RPE layer was isolated from frozen posterior eyeball tissue after penetrating keratoplasty surgery. The tumorigenicity of RPE cells was examined by G-banding and a tumor formation assay in nude mice. Immunogenicity was measured using a one-way mixed lymphocyte reaction (MLR) assay. Dopamine-production in chemically reprogrammed RPE cells was measured by HPLC. Finally, RPE cells were grafted into the brains of monkeys with MPTP-induced PD in order to investigate the potential of such cells treating PD patients in the future. RESULTS: RPE cell lines have been successively established from adult human eye tissues. Such cells can be chemically reprogrammed into dopamine-producing cells in vitro. Moreover, after being grafted into the brain caudate putamen of monkeys with MPTP-induced PD, RPE cells became tyrosine hydroxylase-positive cells, and recipient PD monkeys showed significant improvement of clinical conditions. CONCLUSIONS: This preclinical study using a primate model indicates that human adult RPE cells could be a potential cell source for the treatment of PD in the future.

Mesenchymal origin of multipotent human testis-derived stem cells in human testicular cell cultures.

In contrast to mouse germ cell-derived pluripotent stem cells, the pluripotent state of human testis-derived embryonic stem cell (ESC)-like that spontaneously arise in primary testicular cell cultures remains controversial. Recent studies have shown that these cells closely resemble multipotent mesenchymal stem cells (MSCs), raising the question of their origin and designating these cell populations as multipotent human testis-derived stem cells (mhtSCs) rather than truly ESC-like cells. Here, we evaluate the origin of mhtSCs in vitro by culturing selected testicular cell types. We demonstrate that mhtSCs can be obtained equally efficiently in cultures of pure testicular somatic cells devoid of germ cells. Conversely, cultures with a purified population of germ cells/spermatogonia do not produce any mhtSCs. Based on common molecular characteristics of the somatic starting population and mhtSCs, we conclude that mhtSCs colonies originate from somatic mesenchymal progenitors present in primary testicular cell cultures and do not arise from germ cells undergoing incomplete reprogramming in vitro.

OCT4, SOX2 and NANOG co-regulate glycolysis and participate in somatic induced reprogramming.

OCT4, SOX2 and NANOG (OSN) are the key factors of cell reprogramming, which are involved in the maintenance of stem cell pluripotency. Recently, it has been found that glycolysis plays an important role in the process of somatic-cell-induced reprogramming; however, the synergistic effect of OSN on glycolysis has rarely been reported. In this study, chicken embryonic fibroblasts (CEF) was reprogrammed into induced pluripotent stem cells (iPSCs) by OCT4, SOX2, NANOG and LIN28 reprogramming strategy. RNA-seq showed that chicken iPSCs highly expressed pluripotent genes and the expression of the key genes of glycolysis, such as Hk1, Pfkp and Ldha, was also at a high level, while CEF was much lower. Glycolysis gene expression, glucose uptake and lactate production of CEF and iPSCs were also detected. The results showed that the glycolysis level of iPSCs was higher than that of CEF. ChIP-qPCR showed that SOX2 and NANOG transcription factors were significantly enriched in the promoter regions of Hk1, Pfkp and Ldha, while OCT4 was not. The above results indicated that OCT4, SOX2 and NANOG coordinately regulate glycolysis and participate in somatic-cell-induced reprogramming, thus setting a good foundation for further research on the molecular mechanism of somatic-cell-induced reprogramming. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00530-6.

Tumorigenicity risk of iPSCs in vivo: nip it in the bud.

In 2006, Takahashi and Yamanaka first created induced pluripotent stem cells from mouse fibroblasts via the retroviral introduction of genes encoding the transcription factors Oct3/4, Sox2, Klf44, and c-Myc. Since then, the future clinical application of somatic cell reprogramming technology has become an attractive research topic in the field of regenerative medicine. Of note, considerable interest has been placed in circumventing ethical issues linked to embryonic stem cell research. However, tumorigenicity, immunogenicity, and heterogeneity may hamper attempts to deploy this technology therapeutically. This review highlights the progress aimed at reducing induced pluripotent stem cells tumorigenicity risk and how to assess the safety of induced pluripotent stem cells cell therapy products.

Oncogene-Induced Reprogramming in Acute Lymphoblastic Leukemia: Towards Targeted Therapy of Leukemia-Initiating Cells.

Our understanding of the hierarchical structure of acute leukemia has yet to be fully translated into therapeutic approaches. Indeed, chemotherapy still has to take into account the possibility that leukemia-initiating cells may have a distinct chemosensitivity profile compared to the bulk of the tumor, and therefore are spared by the current treatment, causing the relapse of the disease. Therefore, the identification of the cell-of-origin of leukemia remains a longstanding question and an exciting challenge in cancer research of the last few decades. With a particular focus on acute lymphoblastic leukemia, we present in this review the previous and current concepts exploring the phenotypic, genetic and functional heterogeneity in patients. We also discuss the benefits of using engineered mouse models to explore the early steps of leukemia development and to identify the biological mechanisms driving the emergence of leukemia-initiating cells. Finally, we describe the major prospects for the discovery of new therapeutic strategies that specifically target their aberrant stem cell-like functions.

Sphere-induced reprogramming of RPE cells into dual-potential RPE stem-like cells.

BACKGROUND: The retinal pigment epithelium (RPE) has the potential to regenerate the entire neuroretina upon retinal injury in amphibians. In contrast, this regenerative capacity has been lost in mammals. The reprogramming of differentiated somatic cells into induced pluripotent stem cells (iPSCs) by viral transduction of exogenous stem cell factors has triggered a revolution in regenerative medicine. However, the risks of potential mutation(s) caused by random viral vector insertion in host genomes and tumor formation in recipients hamper its clinical application. One alternative is to immortalize adult stem cells with limited potential or to partially reprogram differentiated somatic cells into progenitor-like cells through non-integration protocols. METHODS: Sphere-induced RPE stem cells (iRPESCs) were generated from adult mouse RPE cells. Their stem cell functionality was studied in a mouse model of retinal degeneration. The molecular mechanism underlying the sphere-induced reprogramming was investigated using microarray and loss-of-function approaches. FINDINGS: We provide evidence that our sphere-induced reprogramming protocol can immortalize and transform mouse RPE cells into iRPESCs with dual potential to differentiate into cells that express either RPE or photoreceptor markers both in vitro and in vivo. When subretinally transplanted into mice with retinal degeneration, iRPESCs can integrate to the RPE and neuroretina, thereby delaying retinal degeneration in the model animals. Our molecular analyses indicate that the Hippo signaling pathway is important in iRPESC reprogramming. INTERPRETATION: The Hippo factor Yap1 is activated in the nuclei of cells at the borders of spheres. The factors Zeb1 and P300 downstream of the Hippo pathway are shown to bind to the promoters of the stemness genes Oct4, Klf4 and Sox2, thereby likely transactivate them to reprogram RPE cells into iRPESCs. FUND: National Natural Science Foundation of China and the National Institute of Health USA.

Stem cells on regenerative and reproductive science in domestic animals.

Stem cells are undifferentiated and self-renewable cells that present new possibilities for both regenerative medicine and the understanding of early mammalian development. Adult multipotent stem cells are already widely used worldwide in human and veterinary medicine, and their therapeutic signalling, particularly with respect to immunomodulation, and their trophic properties have been intensively studied. The derivation of embryonic stem cells (ESCs) from domestic species, however, has been challenging, and the poor results do not reflect the successes obtained in mouse and human experiments. More recently, the generation of induced pluripotent stem cells (iPSCs) via the forced expression of specific transcription factors has been demonstrated in domestic species and has introduced new potentials in regenerative medicine and reproductive science based upon the ability of these cells to differentiate into a variety of cells types in vitro. For example, iPSCs have been differentiated into primordial germ-like cells (PGC-like cells, PGCLs) and functional gametes in mice. The possibility of using iPSCs from domestic species for this purpose would contribute significantly to reproductive technologies, offering unprecedented opportunities to restore fertility, to preserve endangered species and to generate transgenic animals for biomedical applications. Therefore, this review aims to provide an updated overview of adult multipotent stem cells and to discuss new possibilities introduced by the generation of iPSCs in domestic animals, highlighting the possibility of generating gametes in vitro via PGCL induction.