Mitochondrial Permeability Transition in Stem Cells, Development, and Disease.

The mitochondrial permeability transition (mPT) is a process that permits rapid exchange of small molecules across the inner mitochondrial membrane (IMM) and thus plays a vital role in mitochondrial function and cellular signaling. Formation of the pore that mediates this flux is well-documented in injury and disease but its regulation has also emerged as critical to the fate of stem cells during embryonic development. The precise molecular composition of the mPTP has been enigmatic, with far more genetic studies eliminating molecular candidates than confirming them. Rigorous studies in the recent decade have implicated central involvement of the F1Fo ATP synthase, or complex V of the electron transport chain, and continue to confirm a regulatory role for Cyclophilin D (CypD), encoded by Ppif, in modulating the sensitivity of the pore to opening. A host of endogenous molecules have been shown to trigger flux characteristic of mPT, including positive regulators such as calcium ions, reactive oxygen species, inorganic phosphate, and fatty acids. Conductance of the pore has been described as low or high, and reversibility of pore opening appears to correspond with the relative abundance of negative regulators of mPT such as adenine nucleotides, hydrogen ion, and divalent cations that compete for calcium-binding sites in the mPTP. Current models suggest that distinct pores could be responsible for differing reversibility and conductance depending upon cellular context. Indeed, irreversible propagation of mPT inevitably leads to collapse of transmembrane potential, arrest of ATP synthesis, mitochondrial swelling, and cell death. Future studies should clarify ambiguities in mPTP structure and reveal new roles for mPT in dictating specialized cellular functions beyond cell survival that are tied to mitochondrial fitness including stem cell self-renewal and fate. The focus of this review is to describe contemporary models of the mPTP and highlight how pore activity impacts stem cells and development.

RhoA-ROCK competes with YAP to regulate amoeboid breast cancer cell migration in response to lymphatic-like flow.

Lymphatic drainage generates force that induces prostate cancer cell motility via activation of Yes-associated protein (YAP), but whether this response to fluid force is conserved across cancer types is unclear. Here, we show that shear stress corresponding to fluid flow in the initial lymphatics modifies taxis in breast cancer, whereas some cell lines use rapid amoeboid migration behavior in response to fluid flow, a separate subset decrease movement. Positive responders displayed transcriptional profiles characteristic of an amoeboid cell state, which is typical of cells advancing at the edges of neoplastic tumors. Regulation of the HIPPO tumor suppressor pathway and YAP activity also differed between breast subsets and prostate cancer. Although subcellular localization of YAP to the nucleus positively correlated with overall velocity of locomotion, YAP gain- and loss-of-function demonstrates that YAP inhibits breast cancer motility but is outcompeted by other pro-taxis mediators in the context of flow. Specifically, we show that RhoA dictates response to flow. GTPase activity of RhoA, but not Rac1 or Cdc42 Rho family GTPases, is elevated in cells that positively respond to flow and is unchanged in cells that decelerate under flow. Disruption of RhoA or the RhoA effector, Rho-associated kinase (ROCK), blocked shear stress-induced motility. Collectively, these findings identify biomechanical force as a regulator amoeboid cell migration and demonstrate stratification of breast cancer subsets by flow-sensing mechanotransduction pathways.

Biomechanical Regulation of Hematopoietic Stem Cells in the Developing Embryo.

PURPOSE OF REVIEW: The contribution of biomechanical forces to hematopoietic stem cell (HSC) development in the embryo is a relatively nascent area of research. Herein, we address the biomechanics of the endothelial-to-hematopoietic transition (EHT), impact of force on organelles, and signaling triggered by extrinsic forces within the aorta-gonad-mesonephros (AGM), the primary site of HSC emergence. RECENT FINDINGS: Hemogenic endothelial cells undergo carefully orchestrated morphological adaptations during EHT. Moreover, expansion of the stem cell pool during embryogenesis requires HSC extravasation into the circulatory system and transit to the fetal liver, which is regulated by forces generated by blood flow. Findings from other cell types also suggest that forces external to the cell are sensed by the nucleus and mitochondria. Interactions between these organelles and the actin cytoskeleton dictate processes such as cell polarization, extrusion, division, survival, and differentiation. SUMMARY: Despite challenges of measuring and modeling biophysical cues in the embryonic HSC niche, the past decade has revealed critical roles for mechanotransduction in governing HSC fate decisions. Lessons learned from the study of the embryonic hematopoietic niche promise to provide critical insights that could be leveraged for improvement in HSC generation and expansion ex vivo.

Ex Vivo Modeling of Hematopoietic Stem Cell Homing to the Fetal Liver.

Hematopoietic stem cells (HSCs) are used in the clinic to provide life-saving therapies to patients with a variety of hematological malignancies and disorders. Yet, serious deficiencies in our understanding of how HSCs develop and self-renew continue to limit our ability to make this therapy safer and more broadly available to those who have no available donor. Finding ways to expand HSCs and develop alternate sources of HSCs is an urgent priority. In the embryo, a critical transition in development of the blood system requires that newly emergent HSCs from the aorta-gonad-mesonephros (AGM) region migrate to the fetal liver where they aggressively self-renew and expand to numbers sufficient to sustain the adult long term. This process of homing to the fetal liver is orchestrated by intrinsic regulators such as epigenetic modifications to the genome, expression of transcription factors, and adhesion molecule presentation, as well as sensing of extrinsic factors like chemokines, cytokines, and other molecules. Due to technical limitations in manipulating the fetal tissue microenvironment, mechanisms mediating the homing and expansion process remain incompletely understood. Importantly, HSC development is strictly dependent upon forces created by the flow of blood, and current experimental methods make the study of biophysical cues especially challenging. In the protocol presented herein, we address these limitations by designing a biomimetic ex vivo microfluidic model of the fetal liver that enables monitoring of HSC homing to and interaction with fetal liver niches under flow and matrix elasticity conditions typical during embryonic development. This model can be easily customized for the study of key microenvironmental factors and biophysical cues that support HSC homing and expansion.

Mitochondrial Transfer and Regulators of Mesenchymal Stromal Cell Function and Therapeutic Efficacy.

Mesenchymal stromal cell (MSC) metabolism plays a crucial role in the surrounding microenvironment in both normal physiology and pathological conditions. While MSCs predominantly utilize glycolysis in their native hypoxic niche within the bone marrow, new evidence reveals the importance of upregulation in mitochondrial activity in MSC function and differentiation. Mitochondria and mitochondrial regulators such as sirtuins play key roles in MSC homeostasis and differentiation into mature lineages of the bone and hematopoietic niche, including osteoblasts and adipocytes. The metabolic state of MSCs represents a fine balance between the intrinsic needs of the cellular state and constraints imposed by extrinsic conditions. In the context of injury and inflammation, MSCs respond to reactive oxygen species (ROS) and damage-associated molecular patterns (DAMPs), such as damaged mitochondria and mitochondrial products, by donation of their mitochondria to injured cells. Through intercellular mitochondria trafficking, modulation of ROS, and modification of nutrient utilization, endogenous MSCs and MSC therapies are believed to exert protective effects by regulation of cellular metabolism in injured tissues. Similarly, these same mechanisms can be hijacked in malignancy whereby transfer of mitochondria and/or mitochondrial DNA (mtDNA) to cancer cells increases mitochondrial content and enhances oxidative phosphorylation (OXPHOS) to favor proliferation and invasion. The role of MSCs in tumor initiation, growth, and resistance to treatment is debated, but their ability to modify cancer cell metabolism and the metabolic environment suggests that MSCs are centrally poised to alter malignancy. In this review, we describe emerging evidence for adaptations in MSC bioenergetics that orchestrate developmental fate decisions and contribute to cancer progression. We discuss evidence and potential strategies for therapeutic targeting of MSC mitochondria in regenerative medicine and tissue repair. Lastly, we highlight recent progress in understanding the contribution of MSCs to metabolic reprogramming of malignancies and how these alterations can promote immunosuppression and chemoresistance. Better understanding the role of metabolic reprogramming by MSCs in tissue repair and cancer progression promises to broaden treatment options in regenerative medicine and clinical oncology.

Bone marrow stromal cell therapy improves survival after radiation injury but does not restore endogenous hematopoiesis.

The only available option to treat radiation-induced hematopoietic syndrome is allogeneic hematopoietic cell transplantation, a therapy unavailable to many patients undergoing treatment for malignancy, which would also be infeasible in a radiological disaster. Stromal cells serve as critical components of the hematopoietic stem cell niche and are thought to protect hematopoietic cells under stress. Prior studies that have transplanted mesenchymal stromal cells (MSCs) without co-administration of a hematopoietic graft have shown underwhelming rescue of endogenous hematopoiesis and have delivered the cells within 24 h of radiation exposure. Herein, we examine the efficacy of a human bone marrow-derived MSC therapy delivered at 3 h or 30 h in ameliorating radiation-induced hematopoietic syndrome and show that pancytopenia persists despite MSC therapy. Animals exposed to radiation had poorer survival and experienced loss of leukocytes, platelets, and red blood cells. Importantly, mice that received a therapeutic dose of MSCs were significantly less likely to die but experienced equivalent collapse of the hematopoietic system. The cause of the improved survival was unclear, as complete blood counts, splenic and marrow cellularity, numbers and function of hematopoietic stem and progenitor cells, and frequency of niche cells were not significantly improved by MSC therapy. Moreover, human MSCs were not detected in the bone marrow. MSC therapy reduced crypt dropout in the small intestine and promoted elevated expression of growth factors with established roles in gut development and regeneration, including PDGF-A, IGFBP-3, IGFBP-2, and IGF-1. We conclude that MSC therapy improves survival not through overt hematopoietic rescue but by positive impact on other radiosensitive tissues, such as the intestinal mucosa. Collectively, these data reveal that MSCs could be an effective countermeasure in cancer patients and victims of nuclear accidents but that MSCs alone do not significantly accelerate or contribute to recovery of the blood system.

Mechanoregulation in hematopoiesis and hematologic disorders.

PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) are reliant on intrinsic and extrinsic factors for tight control of self-renewal, quiescence, differentiation, and homing. Given the intimate relationship between HSCs and their niche, increasing numbers of studies are examining how biophysical cues in the hematopoietic microenvironment impact HSC functions. RECENT FINDINGS: Numerous mechanosensors are present on hematopoietic cells, including integrins, mechanosensitive ion channels, and primary cilia. Integrin-ligand adhesion, in particular, has been found to be critical for homing and anchoring of HSCs and progenitors in the bone marrow. Integrin-mediated interactions with ligands present on extracellular matrix and endothelial cells are key to establishing long-term engraftment and quiescence of HSCs. Importantly, disruption in the architecture and cellular composition of the bone marrow associated with conditioning regimens and primary myelofibrosis exposes HSCs to a profoundly distinct mechanical environment, with potential implications for progression of hematologic dysfunction and pathologies. SUMMARY: Study of the mechanobiological signals that govern hematopoiesis represents an important future step toward understanding HSC biology in homeostasis, aging, and cancer.

Injury intensifies T cell mediated graft-versus-host disease in a humanized model of traumatic brain injury.

The immune system plays critical roles in promoting tissue repair during recovery from neurotrauma but is also responsible for unchecked inflammation that causes neuronal cell death, systemic stress, and lethal immunodepression. Understanding the immune response to neurotrauma is an urgent priority, yet current models of traumatic brain injury (TBI) inadequately recapitulate the human immune response. Here, we report the first description of a humanized model of TBI and show that TBI places significant stress on the bone marrow. Hematopoietic cells of the marrow are regionally decimated, with evidence pointing to exacerbation of underlying graft-versus-host disease (GVHD) linked to presence of human T cells in the marrow. Despite complexities of the humanized mouse, marrow aplasia caused by TBI could be alleviated by cell therapy with human bone marrow mesenchymal stromal cells (MSCs). We conclude that MSCs could be used to ameliorate syndromes triggered by hypercytokinemia in settings of secondary inflammatory stimulus that upset marrow homeostasis such as TBI. More broadly, this study highlights the importance of understanding how underlying immune disorders including immunodepression, autoimmunity, and GVHD might be intensified by injury.

AIBP-mediated cholesterol efflux instructs hematopoietic stem and progenitor cell fate.

Hypercholesterolemia, the driving force of atherosclerosis, accelerates the expansion and mobilization of hematopoietic stem and progenitor cells (HSPCs). The molecular determinants connecting hypercholesterolemia with hematopoiesis are unclear. Here, we report that a somite-derived prohematopoietic cue, AIBP, orchestrates HSPC emergence from the hemogenic endothelium, a type of specialized endothelium manifesting hematopoietic potential. Mechanistically, AIBP-mediated cholesterol efflux activates endothelial Srebp2, the master transcription factor for cholesterol biosynthesis, which in turn transactivates Notch and promotes HSPC emergence. Srebp2 inhibition impairs hypercholesterolemia-induced HSPC expansion. Srebp2 activation and Notch up-regulation are associated with HSPC expansion in hypercholesterolemic human subjects. Genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq), RNA sequencing (RNA-seq), and assay for transposase-accessible chromatin using sequencing (ATAC-seq) indicate that Srebp2 transregulates Notch pathway genes required for hematopoiesis. Our studies outline an AIBP-regulated Srebp2-dependent paradigm for HSPC emergence in development and HPSC expansion in atherosclerotic cardiovascular disease.

TAZ responds to fluid shear stress to regulate the cell cycle.

Physical forces associated with tumor growth and drainage alter cancer cell invasiveness and metastatic potential. We previously showed that fluid frictional force, or shear stress, typical of lymphatic flow induces YAP1/TAZ activation in prostate cancer cells to promote motility dependent upon YAP1 but not TAZ. Here, we show that shear stress elevates TAZ protein levels and promotes TAZ nuclear localization. Increased TAZ activity drives increased DNA synthesis and induces AMOTL2, ANKRD1, and CTGF gene transcription independently of YAP1. Ectopic expression of constitutively activated TAZ increases expression of these TAZ target genes and promotes cell proliferation of prostate cancer cells. Conversely, silencing of TAZ results in reduced proliferation. Together, our data show that force-induced TAZ regulates signaling that dictates cell division, and suggest that TAZ may govern cellular proliferation of cancer cells traveling through the lymphatics in response to biophysical cues.

A Co-culture Assay to Determine Efficacy of TNF-α Suppression by Biomechanically Induced Human Bone Marrow Mesenchymal Stem Cells.

The beneficial effects of mesenchymal stem cell (MSC)-based cellular therapies are believed to be mediated primarily by the ability odansf MSCs to suppress inflammation associated with chronic or acute injury, infection, autoimmunity, and graft-versus-host disease. To specifically address the effects of frictional force caused by blood flow, or wall shear stress (WSS), on human MSC immunomodulatory function, we have utilized microfluidics to model WSS at the luminal wall of arteries. Anti-inflammatory potency of MSCs was subsequently quantified via measurement of TNF-α production by activated murine splenocytes in co-culture assays. The TNF-α suppression assay serves as a reproducible platform for functional assessment of MSC potency and demonstrates predictive value as a surrogate assay for MSC therapeutic efficacy.

Focal adhesion kinase signaling regulates anti-inflammatory function of bone marrow mesenchymal stromal cells induced by biomechanical force.

Mesenchymal stromal cells (MSCs) have tremendous potential for use in regenerative medicine due to their multipotency and immune cell regulatory functions. Biomimetic physical forces have been shown to direct differentiation and maturation of MSCs in tissue engineering applications; however, the effect of force on immunomodulatory activity of MSCs has been largely overlooked. Here we show in human bone marrow-derived MSCs that wall shear stress (WSS) equivalent to the fluid frictional force present in the adult arterial vasculature significantly enhances expression of four genes that mediate MSC immune regulatory function, PTGS2, HMOX1, IL1RN, and TNFAIP6. Several mechanotransduction pathways are stimulated by WSS, including calcium ion (Ca2+) flux and activation of Akt, MAPK, and focal adhesion kinase (FAK). Inhibition of PI3K-Akt by LY294002 or Ca2+ signaling with chelators, ion channel inhibitors, or Ca2+ free culture conditions failed to attenuate WSS-induced COX2 expression. In contrast, the FAK inhibitor PF-562271 blocked COX2 induction, implicating focal adhesions as critical sensory components upstream of this key immunomodulatory factor. In co-culture assays, WSS preconditioning stimulates MSC anti-inflammatory activity to more potently suppress TNF-α production by activated immune cells, and this improved potency depended upon the ability of FAK to stimulate COX2 induction. Taken together, our data demonstrate that biomechanical force potentiates the reparative and regenerative properties of MSCs through a FAK signaling cascade and highlights the potential for innovative force-based approaches for enhancement in MSC therapeutic efficacy.

Prostaglandin E2 Indicates Therapeutic Efficacy of Mesenchymal Stem Cells in Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) is soon predicted to become the third leading cause of death and disability worldwide. After the primary injury, a complex set of secondary injuries develops hours and days later with prolonged neuroinflammation playing a key role. TBI and other inflammatory conditions are currently being treated in preclinical and clinical trials by a number of cellular therapies. Mesenchymal stem cells (MSC) are of great interest due to their widespread usage, safety, and relative ease to isolate and culture. However, there has been a wide range in efficacy reported using MSC clinically and in preclinical models, likely due to differences in cell preparations and a significant amount of donor variability. In this study, we seek to find a correlation between in vitro activity and in vivo efficacy. We designed assays to explore the responsiveness of MSC to immunological cues to address the immunomodulatory properties of MSC, one of their primary modes of therapeutic activity in TBI. Our results showed intrinsic differences in the immunomodulatory capacity of MSC preparations from different bone marrow and amniotic fluid donors. This difference mirrored the therapeutic capacity of the MSC in an experimental model of TBI, an effect confirmed using siRNA knockdown of COX2 followed by overexpressing COX2. Among the immunomodulatory factors assessed, the therapeutic benefit correlated with the secretion of prostaglandin E2 (PGE2 ) by MSC prior to treatment, suggesting that measurement of PGE2 could be a very useful potency marker to create an index of predicted efficacy for preparations of MSC to treat TBI. Stem Cells 2017;35:1416-1430.

Biomechanical Forces Promote Immune Regulatory Function of Bone Marrow Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are believed to mobilize from the bone marrow in response to inflammation and injury, yet the effects of egress into the vasculature on MSC function are largely unknown. Here we show that wall shear stress (WSS) typical of fluid frictional forces present on the vascular lumen stimulates antioxidant and anti-inflammatory mediators, as well as chemokines capable of immune cell recruitment. WSS specifically promotes signaling through NFκB-COX2-prostaglandin E2 (PGE2 ) to suppress tumor necrosis factor-α (TNF-α) production by activated immune cells. Ex vivo conditioning of MSCs by WSS improved therapeutic efficacy in a rat model of traumatic brain injury, as evidenced by decreased apoptotic and M1-type activated microglia in the hippocampus. These results demonstrate that force provides critical cues to MSCs residing at the vascular interface which influence immunomodulatory and paracrine activity, and suggest the potential therapeutic use of force for MSC functional enhancement. Stem Cells 2017;35:1259-1272.

Fluid shear stress activates YAP1 to promote cancer cell motility.

Mechanical stress is pervasive in egress routes of malignancy, yet the intrinsic effects of force on tumour cells remain poorly understood. Here, we demonstrate that frictional force characteristic of flow in the lymphatics stimulates YAP1 to drive cancer cell migration; whereas intensities of fluid wall shear stress (WSS) typical of venous or arterial flow inhibit taxis. YAP1, but not TAZ, is strictly required for WSS-enhanced cell movement, as blockade of YAP1, TEAD1-4 or the YAP1-TEAD interaction reduces cellular velocity to levels observed without flow. Silencing of TEAD phenocopies loss of YAP1, implicating transcriptional transactivation function in mediating force-enhanced cell migration. WSS dictates expression of a network of YAP1 effectors with executive roles in invasion, chemotaxis and adhesion downstream of the ROCK-LIMK-cofilin signalling axis. Altogether, these data implicate YAP1 as a fluid mechanosensor that functions to regulate genes that promote metastasis.

Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis.

Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo, yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. Here we show that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at embryonic day 9.5, an embryonic stage not previously described to harbor HSCs. Effects on hematopoiesis are mediated in part by a cascade downstream of wall shear stress that involves calcium efflux and stimulation of the prostaglandin E2 (PGE2)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling axis. Blockade of the PGE2-cAMP-PKA pathway in the aorta-gonad-mesonephros (AGM) abolished enhancement in hematopoietic activity. Furthermore, Ncx1 heartbeat mutants, as well as static cultures of AGM, exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB). Similar to flow-exposed cultures, transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution. These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential.

Notch1 acts via Foxc2 to promote definitive hematopoiesis via effects on hemogenic endothelium.

Hematopoietic and vascular development share many common features, including cell surface markers and sites of origin. Recent lineage-tracing studies have established that definitive hematopoietic stem and progenitor cells arise from vascular endothelial-cadherin(+) hemogenic endothelial cells of the aorta-gonad-mesonephros region, but the genetic programs underlying the specification of hemogenic endothelial cells remain poorly defined. Here, we discovered that Notch induction enhances hematopoietic potential and promotes the specification of hemogenic endothelium in differentiating cultures of mouse embryonic stem cells, and we identified Foxc2 as a highly upregulated transcript in the hemogenic endothelial population. Studies in zebrafish and mouse embryos revealed that Foxc2 and its orthologs are required for the proper development of definitive hematopoiesis and function downstream of Notch signaling in the hemogenic endothelium. These data establish a pathway linking Notch signaling to Foxc2 in hemogenic endothelial cells to promote definitive hematopoiesis.

Application of fluid mechanical force to embryonic sources of hemogenic endothelium and hematopoietic stem cells.

During embryonic development, hemodynamic forces caused by blood flow support vascular remodeling, arterialization of luminal endothelium, and hematopoietic stem cell (HSC) emergence. Previously, we reported that fluid shear stress plays a key role in stimulating nitric oxide (NO) signaling in the aorta-gonad-mesonephros (AGM) and is essential for definitive hematopoiesis. We employed a Dynamic Flow System modified from a cone-and-plate assembly to precisely regulate in vitro exposure of AGM cells to a defined pattern of laminar shear stress. Here, we present the design of a microfluidic platform accessible to any research group that requires small cell numbers and allows for recirculation of paracrine signaling factors with minimal damage to nonadherent hematopoietic progenitors and stem cells. We detail the assembly of the microfluidic platform using commercially available components and provide specific guidance in the use of an emerging standard in the measurement of embryonic HSC potential, intravenous neonatal transplantation.

Effect of developmental stage of HSC and recipient on transplant outcomes.

The first hematopoietic stem cells (HSCs) that engraft irradiated adult mice arise in the aorta-gonad-mesonephros (AGM) on embryonic day 11.5 (E11.5). However, at this stage, there is a discrepancy between the apparent frequency of HSCs depicted with imaging and their rarity when measured with limiting dilution transplant. We have attempted to reconcile this difference using neonatal recipients, which are more permissive for embryonic HSC engraftment. We found that embryonic HSCs from E9.5 and E10.5 preferentially engrafted neonates, whereas developmentally mature, definitive HSCs from E14.5 fetal liver or adult bone marrow (BM) more robustly engrafted adults. Neonatal engraftment was enhanced after treating adult BM-derived HSCs with interferon. Adult BM-derived HSCs preferentially homed to the liver in neonatal mice yet showed balanced homing to the liver and spleen in adults. These findings emphasize the functional differences between nascent and mature definitive HSCs.