Mitochondrial permeability transition dictates mitochondrial maturation upon switch in cellular identity of hematopoietic precursors.

The mitochondrial permeability transition pore (mPTP) is a supramolecular channel that regulates exchange of solutes across cristae membranes, with executive roles in mitochondrial function and cell death. The contribution of the mPTP to normal physiology remains debated, although evidence implicates the mPTP in mitochondrial inner membrane remodeling in differentiating progenitor cells. Here, we demonstrate that strict control over mPTP conductance shapes metabolic machinery as cells transit toward hematopoietic identity. Cells undergoing the endothelial-to-hematopoietic transition (EHT) tightly control chief regulatory elements of the mPTP. During EHT, maturing arterial endothelium restricts mPTP activity just prior to hematopoietic commitment. After transition in cellular identity, mPTP conductance is restored. In utero treatment with NIM811, a molecule that blocks sensitization of the mPTP to opening by Cyclophilin D (CypD), amplifies oxidative phosphorylation (OXPHOS) in hematopoietic precursors and increases hematopoiesis in the embryo. Additionally, differentiating pluripotent stem cells (PSCs) acquire greater organization of mitochondrial cristae and hematopoietic activity following knockdown of the CypD gene, Ppif. Conversely, knockdown of Opa1, a GTPase critical for proper cristae architecture, induces cristae irregularity and impairs hematopoiesis. These data elucidate a mechanism that regulates mitochondrial maturation in hematopoietic precursors and underscore a role for the mPTP in the acquisition of hematopoietic fate.

Persistent tailoring of MSC activation through genetic priming.

Mesenchymal stem/stromal cells (MSCs) are an attractive platform for cell therapy due to their safety profile and unique ability to secrete broad arrays of immunomodulatory and regenerative molecules. Yet, MSCs are well known to require preconditioning or priming to boost their therapeutic efficacy. Current priming methods offer limited control over MSC activation, yield transient effects, and often induce expression of pro-inflammatory effectors that can potentiate immunogenicity. Here, we describe a 'genetic priming' method that can both selectively and sustainably boost MSC potency via the controlled expression of the inflammatory-stimulus-responsive transcription factor IRF1 (interferon response factor 1). MSCs engineered to hyper-express IRF1 recapitulate many core responses that are accessed by biochemical priming using the proinflammatory cytokine interferon-γ (IFNγ). This includes the upregulation of anti-inflammatory effector molecules and the potentiation of MSC capacities to suppress T cell activation. However, we show that IRF1-mediated genetic priming is much more persistent than biochemical priming and can circumvent IFNγ-dependent expression of immunogenic MHC class II molecules. Together, the ability to sustainably activate and selectively tailor MSC priming responses creates the possibility of programming MSC activation more comprehensively for therapeutic applications.

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.

Bmi1 Maintains the Self-Renewal Property of Innate-like B Lymphocytes.

The self-renewal ability is a unique property of fetal-derived innate-like B-1a lymphocytes, which survive and function without being replenished by bone marrow (BM) progenitors. However, the mechanism by which IgM-secreting mature B-1a lymphocytes self-renew is poorly understood. In this study, we showed that Bmi1 was critically involved in this process. Although Bmi1 is considered essential for lymphopoiesis, the number of mature conventional B cells was not altered when Bmi1 was deleted in the B cell lineage. In contrast, the number of peritoneal B-1a cells was significantly reduced. Peritoneal cell transfer assays revealed diminished self-renewal ability of Bmi1-deleted B-1a cells, which was restored by additional deletion of Ink4-Arf, the well-known target of Bmi1 Fetal liver cells with B cell-specific Bmi1 deletion failed to repopulate peritoneal B-1a cells, but not other B-2 lymphocytes after transplantation assays, suggesting that Bmi1 may be involved in the developmental process of B-1 progenitors to mature B-1a cells. Although Bmi1 deletion has also been shown to alter the microenvironment for hematopoietic stem cells, fat-associated lymphoid clusters, the reported niche for B-1a cells, were not impaired in Bmi1 -/- mice. RNA expression profiling suggested lysine demethylase 5B (Kdm5b) as another possible target of Bmi1, which was elevated in Bmi1-/- B-1a cells in a stress setting and might repress B-1a cell proliferation. Our work has indicated that Bmi1 plays pivotal roles in self-renewal and maintenance of fetal-derived B-1a cells.

Mesenchymal Stromal Cell Therapeutic Delivery: Translational Challenges to Clinical Application.

For several decades, multipotent mesenchymal stromal cells (MSCs) have been extensively studied for their therapeutic potential across a wide range of diseases. In the preclinical setting, MSCs demonstrate consistent ability to promote tissue healing, down-regulate excessive inflammation and improve outcomes in animal models. Several proposed mechanisms of action have been posited and demonstrated across an array of in vitro models. However, translation into clinical practice has proven considerably more difficult. A number of prominent well-funded late-phase clinical trials have failed, thus calling out for new efforts to optimize product delivery in the clinical setting. In this review, we discuss novel topics critical to the successful translation of MSCs from pre-clinical to clinical applications. In particular, we focus on the major routes of cell delivery, aspects related to hemocompatibility, and potential safety concerns associated with MSC therapy in the different settings.

Hemogenic Endothelial Cells Can Transition to Hematopoietic Stem Cells through a B-1 Lymphocyte-Biased State during Maturation in the Mouse Embryo.

Precursors of hematopoietic stem cells (pre-HSCs) have been identified as intermediate precursors during the maturation process from hemogenic endothelial cells to HSCs in the aorta-gonad-mesonephros (AGM) region of the mouse embryo at embryonic day 10.5. Although pre-HSCs acquire an efficient adult-repopulating ability after ex vivo co-culture, their native hematopoietic capacity remains unknown. Here, we employed direct transplantation assays of CD45-VE-cadherin(VC)+KIT+(V+K+) cells (containing pre-HSCs) into immunodeficient neonatal mice that permit engraftment of embryonic hematopoietic precursors. We found that freshly isolated V+K+ cells exhibited significantly greater B-1 lymphocyte-biased repopulating capacity than multilineage repopulating capacity. Additionally, B cell colony-forming assays demonstrated the predominant B-1 progenitor colony-forming ability of these cells; however, increased B-2 progenitor colony-forming ability emerged after co-culture with Akt-expressing AGM endothelial cells, conditions that support pre-HSC maturation into HSCs. Our studies revealed an unexpected B-1 lymphocyte bias of the V+K+ population and acquisition of B-2 potential during commitment to the HSC fate.

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.

Long-Term Engraftment of ESC-Derived B-1 Progenitor Cells Supports HSC-Independent Lymphopoiesis.

It is generally considered that mouse embryonic stem cell (ESC) differentiation into blood cells in vitro recapitulates yolk sac (YS) hematopoiesis. As such, similar to YS-derived B-progenitors, we demonstrate here that ESC-derived B-progenitors differentiate into B-1 and marginal zone B cells, but not B-2 cells in immunodeficient mice after transplantation. ESC-derived B-1 cells were maintained in the recipients for more than 6 months, secreting natural IgM antibodies in vivo. Gene expression profiling displayed a close relationship between ESC- and YS-derived B-1 progenitors. Because there are no hematopoietic stem cells (HSCs) detectable in our ESC differentiation culture, successful long-term engraftment of ESC-derived functional B-1 cells supports the presence of HSC-independent B-1 cell development.

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.