iPSCs in NK Cell Manufacturing and NKEV Development.

Natural killer (NK) cell immunotherapies for cancer can complement existing T cell therapies while benefiting from advancements already made in the immunotherapy field. For NK cell manufacturing, induced pluripotent stem cells (iPSCs) offer advantages including eliminating donor variation and providing an ideal platform for genome engineering. At the same time, extracellular vesicles (EVs) have become a major research interest, and purified NK cell extracellular vesicles (NKEVs) have been shown to reproduce the key functions of their parent NK cells. NKEVs have the potential to be developed into a standalone therapeutic with reduced complexity and immunogenicity compared to cell therapies. This review explores the role iPSC technology can play in both NK cell manufacturing and NKEV development.

iPSC-Derived Natural Killer Cells for Cancer Immunotherapy.

The discovery of human pluripotent stem cells (PSCs) at the turn of the century opened the door to a new generation of regenerative medicine research. Among PSCs, the donors available for induced pluripotent stem cells (iPSCs) are greatest, providing a potentially universal cell source for all types of cell therapies including cancer immunotherapies using natural killer (NK cells). Unlike primary NK cells, those prepared from iPSCs can be prepared with a homogeneous quality and are easily modified to exert a desired response to tumor cells. There already exist several protocols to genetically modify and differentiate iPSCs into NK cells, and each has its own advantages with regards to immunotherapies. In this short review, we detail the benefits of using iPSCs in NK cell immunotherapies and discuss the challenges that must be overcome before this approach becomes mainstream in the clinic.

Application of induced pluripotent stem cells to primary immunodeficiency diseases.

Primary immunodeficiency diseases (PIDs) are a heterogeneous group of rare immune disorders with genetic causes. Effective treatments using hematopoietic stem cells or pharmaceutical agents have been around for decades. However, for many patients, these treatment options are ineffective, partly because the rarity of these PIDs complicates the diagnosis and therapy. Induced pluripotent stem cells (iPSCs) offer a potential solution to these problems. The proliferative capacity of iPSCs allows for the preparation of a large, stable supply of hematopoietic cells with the same genome as the patient, allowing for new human cell models that can trace cellular abnormalities during the pathogenesis and lead to new drug discovery. PID models using patient iPSCs have been instrumental in identifying deviations in the development or function of several types of immune cells, revealing new molecular targets for experimental therapies. These models are only in their early stages and for the most part have recapitulated results from existing models using animals or primary cells. However, iPSC-based models are being used to study complex diseases of other organs, including those with multigenic causes, suggesting that advances in differentiation processes will expand iPSC-based models to complex PIDs as well.

Considerations in hiPSC-derived cartilage for articular cartilage repair.

BACKGROUND: A lack of cell or tissue sources hampers regenerative medicine for articular cartilage damage. MAIN TEXT: We review and discuss the possible use of pluripotent stem cells as a new source for future clinical use. Human induced pluripotent stem cells (hiPSCs) have several advantages over human embryonic stem cells (hESCs). Methods for the generation of chondrocytes and cartilage from hiPSCs have been developed. To reduce the cost of this regenerative medicine, allogeneic transplantation is preferable. hiPSC-derived cartilage shows low immunogenicity like native cartilage, because the cartilage is avascular and chondrocytes are segregated by the extracellular matrix. In addition, we consider our experience with the aberrant deposition of lipofuscin or melanin on cartilage during the chondrogenic differentiation of hiPSCs. SHORT CONCLUSION: Cartilage generated from allogeneic hiPSC-derived cartilage can be used to repair articular cartilage damage.

Ten years of induced pluripotency: from basic mechanisms to therapeutic applications.

Ten years ago, the discovery that mature somatic cells could be reprogrammed into induced pluripotent stem cells (iPSCs) redefined the stem cell field and brought about a wealth of opportunities for both basic research and clinical applications. To celebrate the tenth anniversary of the discovery, the International Society for Stem Cell Research (ISSCR) and Center for iPS Cell Research and Application (CiRA), Kyoto University, together held the symposium 'Pluripotency: From Basic Science to Therapeutic Applications' in Kyoto, Japan. The three days of lectures examined both the mechanisms and therapeutic applications of iPSC reprogramming. Here we summarize the main findings reported, which are testament to how far the field has come in only a decade, as well as the enormous potential that iPSCs hold for the future.

Reprogramming away from the exhausted T cell state.

Induced pluripotent stem cells (iPSCs) describe somatic cells that have been reprogrammed to the pluripotent state from which they can then be differentiated into any cell type of the body. This ability has tremendous implications on a wide number of medical sciences and applications, including cancer treatments. In many cancer patients, tumor infiltrating lymphocytes (TILs) have reached an exhausted state and are unable to exert effector function despite detecting and localizing at the tumor. Although the isolation, ex vivo expansion and transplantation of TILs is effective in a significant group of patients, too many patients do not respond positively to this treatment, in part because the expanded TIL population does not include a sufficient number of cells with the naïve or memory phenotype. Cell reprogramming using iPSC technologies aims to overcome this problem by returning TILs to the pluripotent state from which they can be differentiated into a heterogeneous population of T cells that are best suited to combat the tumor.

Strain through the neck linker ensures processive runs: a DNA-kinesin hybrid nanomachine study.

The motor protein kinesin has two heads and walks along microtubules processively using energy derived from ATP. However, how kinesin heads are coordinated to generate processive movement remains elusive. Here we created a hybrid nanomachine (DNA-kinesin) using DNA as the skeletal structure and kinesin as the functional module. Single molecule imaging of DNA-kinesin hybrid allowed us to evaluate the effects of both connect position of the heads (N, C-terminal or Mid position) and sub-nanometer changes in the distance between the two heads on motility. Our results show that although the native structure of kinesin is not essential for processive movement, it is the most efficient. Furthermore, forward bias by the power stroke of the neck linker, a 13-amino-acid chain positioned at the C-terminus of the head, and internal strain applied to the rear of the head through the neck linker are crucial for the processive movement. Results also show that the internal strain coordinates both heads to prevent simultaneous detachment from the microtubules. Thus, the inter-head coordination through the neck linker facilitates long-distance walking.

Myosin heavy chain isoform expression regulates shortening velocity in smooth muscle: studies using an SMB KO mouse line.

The kinetics of smooth muscle are thought to be partially determined by the level of the expression of the 7 amino acid insert, SMB, in the myosin heavy chain, as SMB is generally expressed at higher levels in faster smooth muscle. In this study, we determined the role of this insert on shortening velocity and force regeneration following rapid reduction in muscle length (k(step)) in bladder tissue from a transgenic mouse line expressing the insert at three different levels: wild type (WT, +/+, SMB/SMB), an SMA homozygous type (SMB KO, -/-), and a heterozygous type (+/-, SMB/SMA). Smooth muscle from +/+ bladder shorten faster than both the +/- and -/- bladder smooth muscle when activated with Ca2+, consistent with SMB determining the shortening velocity of smooth muscle. The addition of Pi to the fully activated skinned bladder strips did not affect the rate of shortening for either the +/+ or -/- bladder types but did significantly decrease the rate of shortening for the +/- type. In contrast, the addition of ADP to fully Ca2+ activated bladder strips increased the rate of shortening for all three bladder types. However after thiophosphorylation, ADP slowed the shortening velocity. These data are consistent with shortening velocity being determined by the level of activation (or crossbridge attachment) in smooth muscle. The rates of force regeneration according to the k(step) protocol showed no differences between bladder types and also proved insensitive to either Pi or ADP. These data suggest that the rates of force regeneration were determined not only by the kinetics of the crossbridge cycle, but also by factors outside the contractile apparatus.

The kinetic properties of smooth muscle: how a little extra weight makes myosin faster.

The contractile properties of smooth muscle (SM) are often described as fast and slow, but the molecular basis for the diversity in contractile properties has yet to be fully elucidated. Studies have shown that the differences in the contractile parameters are seen at the level of the contractile proteins. Experiments have implicated both the splicing of the SM myosin heavy chain (MHC) and the SM myosin essential myosin light chain as possible molecular determinants of the contractile properties of SM. This communication will focus on the role of the 7 aa insert in the smooth muscle MHC in determining the contractile properties of SM and the possible mechanism by which this insert could alter the kinetics of the SM actomyosin ATPase.

The smooth muscle myosin seven amino acid heavy chain insert's kinetic role in the crossbridge cycle for mouse bladder.

The seven amino acid insert in the smooth muscle myosin heavy chain is thought to regulate the kinetics of contraction, contributing to the differences between fast and slow smooth muscle. The effects of this insert on force and stiffness were determined in bladder tissue of a transgenic mouse line expressing the insert SMB at one of three levels: an SMB wild type (+/+), an SMA homozygous type (-/-) and a heterozygous type (+/-). For skinned muscle, an increase in MgADP or inorganic phosphate (Pi) should shift the distribution of crossbridges in the actomyosin ATPase (AMATPase) to increase the relative population of the crossbridge state prior to ADP release and Pi release, respectively. Exogenous ADP increased force and stiffness in a manner consistent with increasing the Ca2+ concentration in both the +/+ and +/- mouse types. However, the -/- type showed a significantly greater increase in force than in stiffness suggesting that immediately prior to ADP release, the AMATPase either has an additional force producing isomerization state or a slower ADP dissociation rate for the -/- type compared to the +/+ or +/- types. Exogenous Pi led to a significantly greater decrease in stiffness than in force for all three mouse types suggesting that there is a force producing state prior to Pi release. In addition, the increase in Pi showed similar changes in the +/+ and -/- types whereas in the +/- type the decreases in both force and stiffness were greater than the other two mouse types indicating that the insert can affect the cooperativity between myosin heads. In conclusion, the seven amino acid insert modulates the kinetics and/or states of the AMATPase, which could lead to differences in the kinetics of contraction between fast and slow smooth muscle.