Rapid Differentiation of Human Embryonic Stem Cells into Testosterone-Producing Leydig Cell-Like Cells In vitro.

BACKGROUND: Leydig cells (LCs) are testicular somatic cells that are the major producers of testosterone in males. Testosterone is essential for male physiology and reproduction. Reduced testosterone levels lead to hypogonadism and are associated with diverse pathologies, such as neuronal dysfunction, cardiovascular disease, and metabolic syndrome. LC transplantation is a promising therapy for hypogonadism; however, the number of LCs in the testis is very rare and they do not proliferate in vitro. Therefore, there is a need for an alternative source of LCs. METHODS: To develop a safer, simple, and rapid strategy to generate human LC-like cells (LLCs) from stem cells, we first performed preliminary tests under different conditions for the induction of LLCs from human CD34/CD73 double positive-testis-derived stem cells (HTSCs). Based on the embryological sequence of events, we suggested a 3-step strategy for the differentiation of human ESCs into LLCs. We generated the mesendoderm in the first stage and intermediate mesoderm (IM) in the second stage and optimized the conditions for differentiation of IM into LLCs by comparing the secreted testosterone levels of each group. RESULTS: HTSCs and human embryonic stem cells can be directly differentiated into LLCs by defined molecular compounds within a short period. Human ESC-derived LLCs can secrete testosterone and express steroidogenic markers. CONCLUSION: We developed a rapid and efficient protocol for the production of LLCs from stem cells using defined molecular compounds. These findings provide a new therapeutic cell source for male hypogonadism.

In Vitro Derivation of Functional Sertoli-Like Cells from Mouse Embryonic Stem Cells.

Sertoli cells (SCs) in the mammalian testes are well known as supporting cells of spermatogenesis, but have recently become an attractive source of cell therapy because of their capacity for immune modulation and trophic effects. In order to increase their applicable efficacy, we demonstrate a novel differentiation method for mouse embryonic stem cell (ESC)-derived Sertoli-like cells (SLCs) via the intermediate mesoderm (IM). We show that IM derived from an induction of 6 days expressed markers such as Wt1, Lhx1, Pax2 and Osr1, and that a sequential induction of 6 days resulted in ESC-SLCs. The SLCs expressed their marker genes ( Sf1, Sox9, Gata4, Wt1, Fshr and Scf), but the pluripotency-marker gene Oct4 was decreased. After sorting by FSHR expression, high-purity (> 90%) SLCs were collected that showed distinct characteristics of SCs such as high phagocytic and immune modulation activities as well as the expression of immune-related genes. In addition, when transplanted into the seminiferous tubule of busulfan-treated mice, SLCs re-located and were maintained in the basal region of the tubule. These results demonstrated that our robust sequential differentiation system produced functional SLCs from mouse ESCs in vitro.

Fetal bovine serum-free cryopreservation methods for clinical banking of human adipose-derived stem cells.

The use of fetal bovine serum (FBS) as a cryopreservation supplement is not suitable for the banking of mesenchymal stem cells (MSCs) due to the risk of transmission of disease as well as xenogeneic immune reactions in the transplanted host. Here, we investigated if human serum albumin (HSA), human serum (HS), or knockout serum replacement (KSR) can replace FBS for the cryopreservation of MSCs. In addition, we examined the characteristics of MSCs after multiple rounds of cryopreservation. Human adipose-derived stem cells (ASCs) cryopreserved with three FBS replacements, 9% HSA, 90% HS, or 90% KSR, in combination with 10% dimethyl sulfoxide (Me2SO) maintained stem cell properties including growth, immunophenotypes, gene expression patterns, and the potential to differentiate into adipogenic, osteogenic, and chondrogenic lineages, similar to ASCs frozen with FBS. Moreover, the immunophenotype, gene expression, and differentiation capabilities of ASCs were not altered by up to four freeze-thaw cycles. However, the performance of three or four freeze-thaw cycles significantly reduced the proliferation ability of ASCs, as indicated by the longer population doubling time and reduced colony-forming unit-fibroblast frequency. Together, our results suggest that HSA, HS, or KSR can replace FBS for the cryopreservation of ASCs, without altering their stemness, and should be processed with no more than two freeze-thaw cycles for clinical approaches.

Transplantation of insulin-secreting cells differentiated from human adipose tissue-derived stem cells into type 2 diabetes mice.

Currently, there are limited ways to preserve or recover insulin secretory capacity in human pancreas. We evaluated the efficacy of cell therapy using insulin-secreting cells differentiated from human eyelid adipose tissue-derived stem cells (hEAs) into type 2 diabetes mice. After differentiating hEAs into insulin-secreting cells (hEA-ISCs) in vitro, cells were transplanted into a type 2 diabetes mouse model. Serum levels of glucose, insulin and c-peptide were measured, and changes of metabolism and inflammation were assessed in mice that received undifferentiated hEAs (UDC group), differentiated hEA-ISCs (DC group), or sham operation (sham group). Human gene expression and immunohistochemical analysis were done. DC group mice showed improved glucose level, and survival up to 60 days compared to those of UDC and sham group. Significantly increased levels of human insulin and c-peptide were detected in sera of DC mice. RT-PCR and immunohistochemical analysis showed human gene expression and the presence of human cells in kidneys of DC mice. When compared to sham mice, DC mice exhibited lower levels of IL-6, triglyceride and free fatty acids as the control mice. Transplantation of hEA-ISCs lowered blood glucose level in type 2 diabetes mice by increasing circulating insulin level, and ameliorating metabolic parameters including IL-6.

Isolation and characterization of novel, highly proliferative human CD34/CD73-double-positive testis-derived stem cells for cell therapy.

Human adult stem cells are a readily available multipotent cell source that can be used in regenerative medicine. Despite many advantages, including low tumorigenicity, their rapid senescence and limited plasticity have curtailed their use in cell-based therapies. In this study, we isolated CD34/CD73-double-positive (CD34(+)/CD73(+)) testicular stromal cells (HTSCs) and found that the expression of CD34 was closely related to the cells' stemness and proliferation. The CD34(+)/CD73(+) cells grew in vitro for an extended period of time, yielding a multitude of cells (5.6×10(16) cells) without forming tumors in vivo. They also differentiated into all three germ layer lineages both in vitro and in vivo, produced cartilage more efficiently compared to bone marrow stem cells and, importantly, restored erectile function in a cavernous nerve crush injury rat model. Thus, these HTSCs may represent a promising new autologous cell source for clinical use.

Insulin-secreting cells from human eyelid-derived stem cells alleviate type I diabetes in immunocompetent mice.

Various attempts have been made to develop stem cell-based therapy to alleviate type I diabetes using animal models. However, it has been a question whether human insulin produced from explanted cells is solely responsible for the normoglycemia of diabetic animals. In this study, we isolated neural crest-like stem cells from the human eyelid fat and examined their therapeutic potentials for diabetes. The human eyelid adipose-derived stem cells (HEACs) displayed characteristics of neural crest cells. Using a two-step culture condition combined with nicotinamide, activin, and/or GLP-1, we differentiated HEACs into insulin-secreting cells and examined in vivo effects of differentiated cells by transplantation experiments. Following differentiation in vitro, HEACs released insulin and c-peptide in a glucose-dependent manner. Upon their transplantation under kidney capsules of streptozotocin-treated immunocompetent mice, we observed normalization of hyperglycemia in 10 of 20 recipient mice until sacrifice after 2 months. Only the human, but not the mouse, insulin and c-peptide were detected in the blood of recipient mice. Removal of the kidneys transplanted with HEACs resulted in a sharp increase of blood glucose level. Removed kidney tissues showed distinct expression of various human genes including insulin, and colocalization of the human insulin and the human nuclear protein in many cells. However, they showed diminished or null expression of some immune-related genes. In conclusion, human insulin alone produced from eyelid-derived stem cells following differentiation into insulin-secreting cells and transplantation could normalize type I diabetes in mice.