Stem Cell-derived Progeny Research Articles

Systematic transcriptome-based comparison of cellular adaptive stress response activation networks in hepatic stem cell-derived progeny and primary human hepatocytes

Various adaptive cellular stress response pathways are critical in the pathophysiology of liver disease and drug-induced liver injury. Human-induced pluripotent stem cell (hiPSC)-derived hepatocyte-like cells (HLCs) provide a promising tool to study cellular stress response pathways, but in this context there is limited insight on how HLCs compare to other in vitro liver models. Here, we systematically compared the transcriptomic profiles upon chemical activation in HLCs, hiPSC, primary human hepatocytes (PHH) and HepG2 liver cancer cells. We used targeted RNA-sequencing to map concentration transcriptional response using benchmark concentration modeling for the various stress responses in the different test systems. We found that HLCs are very sensitive towards oxidative stress and inflammation conditions as corresponding genes were activated at over 3 fold lower concentrations of the corresponding pathway inducing compounds as compared to PHH. PHH were the most sensitive model when studying UPR related effects. Due to the non-proliferative nature of PHH and HLCs, these do not pose a good/sensitive model to pick up DNA damage responses, while hiPSC and HepG2 were more sensitive in these conditions. We envision that this study contributes to a better understanding on how HLCs can contribute to the assessment of cell physiological stress response activation to predict hepatotoxic events.

Engineered Livers for Infectious Diseases.

Engineered liver systems come in a variety of platform models, from 2-dimensional cocultures of primary human hepatocytes and stem cell–derived progeny, to 3-dimensional organoids and humanized mice. Because of the species-specificity of many human hepatropic pathogens, these engineered systems have been essential tools for biologic discovery and therapeutic agent development in the context of liver-dependent infectious diseases. Although improvement of existing models is always beneficial, and the addition of a robust immune component is a particular need, at present, considerable progress has been made using this combination of research platforms. We highlight advances in the study of hepatitis B and C viruses and malaria-causing Plasmodium falciparum and Plasmodium vivax parasites, and underscore the importance of pairing the most appropriate model system and readout modality with the particular experimental question at hand, without always requiring a platform that recapitulates human physiology in its entirety.

Abstract 3765: IND-Enabling GLP study to support a clinical trial of dual adoptive cell therapy combining stem cells and T cells engineered with an NY-ESO-1 TCR

Abstract T cell receptor (TCR) engineered adoptive T cell transfer (ACT) has shown remarkable antitumor efficacy in several clinical trials. However, low persistence of modified cells limits long-term clinical responses. To overcome this hurdle, we propose a clinical trial co-administering genetically modified T cells and stem cells both expressing an NY-ESO-1 TCR such that the engrafted stem cells generate a source for constant renewal of modified T cells. Here we report a pre-clinical IND-enabling study performed at UCLA under Good Laboratory Practice (GLP) compliance to assess whether co-administration impacts (I) safety; (II) engraftment and cell lineage differentiation of gene modified stem cells; and (III) persistence of adoptively transferred T cells and stem cell-derived progeny. We performed 12 optimization studies to define the optimal conditions for TCR gene modified ACT and TCR gene modified hematopoietic stem cell (HSC) bone marrow transplantation (BMT). Sixty-four HLA-A2/kb transgenic mice were myelodepleted and received syngeneic BMT with Lineage depleted bone marrow (Lin-) cells transduced with the LV-NYESO-1 TCR/sr39TK and ACT with T cells transduced with the RV-NYESO-1 TCR. Control groups were as follows: untreated mice, mice receiving mock transduced Lin- cells and T cells, mice receiving transduced Lin- cells and mock transduced T cells, and mice receiving mock transduced Lin- cells and transduced T cells (n = 16 per group). Overall survival at 3 months was 87.5%; no significant differences in survival were observed among cohorts. After BMT we observed a decrease in body weight, elevation in creatinine kinase and transaminases, and gonadal germ cell ablation in all cohorts. Three months after BMT, all blood cell lineages were reconstituted in surviving mice. Using digital droplet PCR and flow cytometry, we confirmed that transduced stem cells engrafted and their progeny persisted long term. In the bone marrow, NY-ESO-1 TCR was expressed intracellularly among progenitor cells (Lin-, LSK and HSC) as well as all hematopoietic cell lineages within the spleen (CD8+ T cells, CD4+ T cells, NKT cells, B cells and granulocytes). Co-administration with gene modified T cells and stem cells did not affect engraftment, cell lineage differentiation or persistence of the gene modified stem cells. Moreover, co-administration with stem cells did not affect persistence of adoptively transferred T cells. These data demonstrate that 1) NY-ESO-1 TCR genetically modified stem cells engraft and differentiate into all hematopoietic cell lineage progeny, which persists at 3 months; 2) adoptively transferred NY-ESO-1 TCR T cells persist at 3 months; 3) co-administration of stem cells and T cells genetically modified to express an NY-ESO-1 TCR is safe and does not negatively impact stem cell engraftment, lineage differentiation and progeny persistence or T cell persistence. Citation Format: Cristina Puig-Saus, Giulia Parisi, Paige Krystofinski, Angel Garcia-Diaz, Salemiz Sandoval, James McCabe, Ruixue Zhang, Gardenia Cheung-Lau, Nhat Truong, Justin Saco, Sara Komenan, Agustin Vega-Crespo, Mignonette H Macabali, Begoña Comin-Anduix, Beata Berent-Maoz, Donald Kohn, Paula Kaplan-Lefko, Antoni Ribas. IND-Enabling GLP study to support a clinical trial of dual adoptive cell therapy combining stem cells and T cells engineered with an NY-ESO-1 TCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3765. doi:10.1158/1538-7445.AM2017-3765

Keratin 79 identifies a novel population of migratory epithelial cells that initiates hair canal morphogenesis and regeneration

The formation of epithelial tubes underlies the development of diverse organs. In the skin, hair follicles resemble tube-like structures with lumens that are generated through poorly understood cellular rearrangements. Here, we show that creation of the hair follicle lumen is mediated by early outward movement of keratinocytes from within the cores of developing hair buds. These migratory keratinocytes express keratin 79 (K79) and stream out of the hair germ and into the epidermis prior to lumen formation in the embryo. Remarkably, this process is recapitulated during hair regeneration in the adult mouse, when K79+ cells migrate out of the reactivated secondary hair germ prior to formation of a new hair canal. During homeostasis, K79+ cells line the hair follicle infundibulum, a domain we show to be multilayered, biochemically distinct and maintained by Lrig1+ stem cell-derived progeny. Upward movement of these cells sustains the infundibulum, while perturbation of this domain during acne progression is often accompanied by loss of K79. Our findings uncover previously unappreciated long-distance cell movements throughout the life cycle of the hair follicle, and suggest a novel mechanism by which the follicle generates its hollow core through outward cell migration.

Keratin 79 identifies a novel population of migratory epithelial cells that initiates hair canal morphogenesis and regeneration

The formation of epithelial tubes underlies the development of diverse organs. In the skin, hair follicles resemble tube-like structures with lumens that are generated through poorly understood cellular rearrangements. Here, we show that creation of the hair follicle lumen is mediated by early outward movement of keratinocytes from within the cores of developing hair buds. These migratory keratinocytes express keratin 79 (K79) and stream out of the hair germ and into the epidermis prior to lumen formation in the embryo. Remarkably, this process is recapitulated during hair regeneration in the adult mouse, when K79(+) cells migrate out of the reactivated secondary hair germ prior to formation of a new hair canal. During homeostasis, K79(+) cells line the hair follicle infundibulum, a domain we show to be multilayered, biochemically distinct and maintained by Lrig1(+) stem cell-derived progeny. Upward movement of these cells sustains the infundibulum, while perturbation of this domain during acne progression is often accompanied by loss of K79. Our findings uncover previously unappreciated long-distance cell movements throughout the life cycle of the hair follicle, and suggest a novel mechanism by which the follicle generates its hollow core through outward cell migration.

Mass Production of Stem Cell-Derived Progeny in Bioreactors

Stem cells, including mesenchymal stem cells (MSCs) and pluripotent stem cells (PSCs), have shown great potential for various biomedical applications including drug discovery, disease modeling, and tissue engineering. Especially, the discovery of induced pluripotent stem cells (iPSCs) with similar characteristics to embryonic stem cells (ESCs) opens a new era for stem cell research and transplantations. Bioprocess engineering provides a platform to generate a controlled microenvironment that could potentially recreate a stem cell niche in view of promoting stem cell proliferation or the lineage-specific differentiation.

InVERT molding for scalable control of tissue microarchitecture

Complex tissues contain multiple cell types that are hierarchically organized within morphologically and functionally distinct compartments. Construction of engineered tissues with optimized tissue architecture has been limited by tissue fabrication techniques, which do not enable versatile microscale organization of multiple cell types in tissues of size adequate for physiologic studies and tissue therapies. Here we present an ‘Intaglio-Void/Embed-Relief Topographic (InVERT) molding’ method for microscale organization of many cell types, including induced pluripotent stem cell (iPS)-derived progeny, within a variety of synthetic and natural extracellular matrices and across tissues of sizes appropriate for in vitro, pre-clinical, and clinical studies. We demonstrate that compartmental placement of non-parenchymal cells relative to primary or iPS-derived hepatocytes, compartment microstructure, and cellular composition modulate hepatic functions. Configurations found to sustain physiologic function in vitro also result in survival and function in mice for at least four weeks, demonstrating the importance of architectural optimization prior to implantation.

Investigating the bona fide differentiation capacity of human pluripotent stem cells

Investigating the bona fide differentiation capacity of human pluripotent stem cells

Proliferation of mouse embryonic stem cell progeny and the spontaneous contractile activity of cardiomyocytes are affected by microtopography

The niche in which stem cells reside and differentiate is a complex physicochemical microenvironment that regulates cell function. The role played by three-dimensional physical contours was studied on cell progeny derived from mouse embryonic stem cells using microtopographies created on PDMS (poly-dimethyl-siloxane) membranes. While markers of differentiation were not affected, the proliferation of heterogeneous mouse embryonic stem cell-derived progeny was attenuated by 15 microm-, but not 5 microm-high microprojections. This reduction was reversed by Rho kinase and myosin light chain kinase inhibition, which diminishes the tension generating ability of stress fibers. Purified cardiomyocytes derived from embryonic stem cells also showed significant blunting of proliferation and increased beating rates compared with cells grown on flat substrates. Thus, proliferation of stem cell-derived progeny appears to be regulated by microtopography through tension-generation of contractility in the third-dimension. These results emphasize the importance of topographic cues in the modulation of stem cell progeny behavior.

Progenitor cell trafficking in the vascular wall.

Adult endothelial as well as smooth muscle progenitor cells are engaged in the complex pathophysiology of atherosclerosis including primary remodeling with development and progression of atherosclerotic plaques as well as secondary complications associated with ischemia, endothelial damage, neointimal growth and transplant arteriosclerosis. These adult vascular precursor cells correspond to similar embryonic stem cell-derived progeny and are primarily located in bone marrow and peripheral blood. Recently, specific investigation on their recruitment emerged as a novel fundamental in the pathogenesis of arterial remodeling, plaque stability and angiogenesis. This multifaceted process of mobilization and homing is regulated by numerous chemokines, adhesion molecules and growth factors that guide and control the trafficking of vascular progenitor cells to the arterial wall after injury or during ischemia.

Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat

Self-renewal of spermatogonial stem cells (SSCs) is the foundation for maintenance of spermatogenesis throughout life in males and for continuation of a species. The molecular mechanism underlying stem cell self-renewal is a fundamental question in stem cell biology. Recently, we identified growth factors necessary for self-renewal of mouse SSCs and established a serum-free culture system for their proliferation in vitro. To determine whether the stimulatory signals for SSC replication are conserved among different species, we extended the culture system to rat SSCs. Initially, a method to assess in vitro expansion of SSCs was developed by using flow cytometric analysis, and, subsequently, we found that a combination of glial cell line-derived neurotrophic factor, soluble glial cell line-derived neurotrophic factor-family receptor alpha-1 and basic fibroblast growth factor supports proliferation of rat SSCs. When cultured with the three factors, stem cells proliferated continuously for >7 months, and transplantation of the cultured SSCs to recipient rats generated donor stem cell-derived progeny, demonstrating that the cultured stem cells are normal. The growth factor requirement for replication of rat SSCs is identical to that of mouse; therefore, the signaling factors for SSC self-renewal are conserved in these two species. Because SSCs from many mammals, including human, can replicate in mouse seminiferous tubules after transplantation, the growth factors required for SSC self-renewal may be conserved among many different species. Furthermore, development of a long-term culture system for rat SSCs has established a foundation for germ-line modification of the rat by gene targeting technology.

Assisted reproductive technologies in rhesus macaques

The assisted reproductive technologies (ARTs) have been used in the production of rhesus monkey offspring at the Oregon National Primate Research Center (ONPRC) and that experience is summarized here. Additionally these technologies serve as a source of oocytes/embryos for monozygotic twinning, embryonic stem (ES) cell derivation and cloning. High fertilization efficiencies were realized with conventional insemination or following the use of intracytoplasmic sperm injection (ICSI) and approximately 50% of the resulting embryos grew in vitro to blastocysts. Both fresh and frozen sperm were employed in fertilization by ICSI and the resulting embryos could be low temperature stored for subsequent thawing and transfer when a synchronized recipient female was available or after shipment to another facility. Following the transfer of up to 3 embryos, an overall pregnancy rate of 30% was achieved with increasing rates dependent upon the number of embryos transferred. Singleton pregnancy outcomes following the transfer of ART produced embryos were similar to those observed in a control group of animals in the timed mated breeding colony at ONPRC. ICSI produced embryos were used in efforts to create monozygotic twins by blastomere separation or blastocyst splitting. While pregnancies were achieved following the transfer of demi-embryos, only one was a twin and it was lost to spontaneous abortion. ICSI produced embryos have also served as the source of blastocysts for the derivation of embryonic stem cells. These pluripotent cells hold potential for cell based therapies and we consider the monkey an important translational model in which to evaluate safety, efficacy and feasibility of regenerative medicine approaches based on the transplantation of stem cell-derived progeny. Finally, efforts to produce genetically-identical monkeys by nuclear transfer have been briefly summarized.

Hair follicle stem cells.

The workshop on Hair Follicle Stem Cells brought together investigators who have used a variety of approaches to try to understand the biology of follicular epithelial stem cells, and the role that these cells play in regulating the hair cycle. One of the main concepts to emerge from this workshop is that follicular epithelial stem cells are multipotent, capable of giving rise not only to all the cell types of the hair, but also to the epidermis and the sebaceous gland. Furthermore, such multipotent stem cells may represent the ultimate epidermal stem cell. Another example of epithelial stem cell and transit amplifying cell plasticity, was the demonstration that adult corneal epithelium, under the influence of embryonic skin dermis could form an epidermis as well as hair follicles. With regards to the location of follicular epithelial stem cells, immunohistochemical and ultrastructural data was presented, indicating that cells with stem cell attributes were localized to the prominent bulge region of developing human fetal hair follicles. Finally, a new notion was put forth concerning the roles that the bulge-located stem cells and the hair germ cells played with respect to the hair cycle.

Cross-species bone marrow transplantation: evidence for tolerance induction, stem cell engraftment, and maturation of T lymphocytes in a xenogeneic stromal environment (rat----mouse).

Transplantation of untreated F344 rat bone marrow into irradiated B10 mouse recipients (non-TCD F344----B10) to produce fully xenogeneic chimeras resulted in stable xenogeneic lymphoid chimerism, ranging from 82% to 97% rat. Survival of animals was excellent, without evidence for GVH disease. The specificity of tolerance which resulted was highly donor-specific; MHC disparate third party mouse and rat skin grafts were promptly rejected while donor-specific F344 grafts were significantly prolonged (MST greater than 130 days). Multi-lineage rat stem cell-derived progeny including lymphoid cells (T- and B-lymphocytes), myeloid cells, erythrocytes, platelets, and natural killer (NK) cells were present in the fully xenogenic chimeras up to 7 months after bone marrow transplantation. Immature rat T-lymphocytes matured and acquired the alpha/beta T-cell receptor in the thymus of chimeras in a pattern similar to normal rat controls, suggesting that immature T-lymphocytes of rat origin could interact with the murine xenogeneic thymic stroma to undergo normal maturation and differentiation. This model may be useful to study the mechanisms responsible for the induction and maintenance of donor-specific transplantation tolerance across a species barrier.