Zeb1 Regulates the Function of Lympho-Myeloid Primed Progenitors after Transplantation.

Zeb1, a zinc finger E-box binding homeobox epithelial-mesenchymal (EMT) transcription factor, acts as a critical regulator of hematopoietic stem cell (HSC) self-renewal and multi-lineage differentiation. Whether Zeb1 directly regulates the function of multi-potent progenitors primed for hematopoietic lineage commitment remains ill defined. By using an inducible Mx-1 Cre conditional mouse model where Zeb1 was genetically engineered to be deficient in the adult hematopoietic system (hereafter Zeb1-/-), we found that the absolute cell number of immunophenotypically defined lympho-myeloid primed progenitors (LMPPs) from Zeb1-/- mice was reduced. Myeloid- and lymphoid-biased HSCs in Zeb1-/- mice were unchanged, implying that defective LMPP generation from Zeb1-/- mice was not directly caused by an imbalance of lineage-biased HSCs. Functional analysis of LMPP from Zeb1-/- mice, as judged by competitive transplantation, revealed an overall reduction in engraftment to hematopoietic organs over 4 weeks, which correlated with minimal T-cell engraftment, reduced B-cell and monocyte/macrophage engraftment, and unperturbed granulocyte engraftment. Thus, Zeb1 regulates LMPP differentiation potential to select lympho-myeloid lineages in the context of transplantation.

An alginate-based encapsulation system for delivery of therapeutic cells to the CNS.

Treatment options for neurodegenerative conditions such as Parkinson's disease have included the delivery of cells which release dopamine or neurotrophic factors to the brain. Here, we report the development of a novel approach for protecting cells after implantation into the central nervous system (CNS), by developing dual-layer alginate beads that encapsulate therapeutic cells and release an immunomodulatory compound in a sustained manner. An optimal alginate formulation was selected with a view to providing a sustained physical barrier between engrafted cells and host tissue, enabling exchange of small molecules while blocking components of the host immune response. In addition, a potent immunosuppressant, FK506, was incorporated into the outer layer of alginate beads using electrosprayed poly-ε-caprolactone core-shell nanoparticles with prolonged release profiles. The stiffness, porosity, stability and ability of the alginate beads to support and protect encapsulated SH-SY5Y cells was demonstrated, and the release profile of FK506 and its effect on T-cell proliferation in vitro was characterized. Collectively, our results indicate this multi-layer encapsulation technology has the potential to be suitable for use in CNS cell delivery, to protect implanted cells from host immune responses whilst providing permeability to nutrients and released therapeutic molecules.

Zeb1 modulates hematopoietic stem cell fates required for suppressing acute myeloid leukemia.

Zeb1, a zinc finger E-box binding homeobox epithelial-mesenchymal transition (EMT) transcription factor, confers properties of "stemness," such as self-renewal, in cancer. Yet little is known about the function of Zeb1 in adult stem cells. Here, we used the hematopoietic system as a well-established paradigm of stem cell biology to evaluate Zeb1-mediated regulation of adult stem cells. We employed a conditional genetic approach using the Mx1-Cre system to specifically knock out (KO) Zeb1 in adult hematopoietic stem cells (HSCs) and their downstream progeny. Acute genetic deletion of Zeb1 led to rapid-onset thymic atrophy and apoptosis-driven loss of thymocytes and T cells. A profound cell-autonomous self-renewal defect and multilineage differentiation block were observed in Zeb1-KO HSCs. Loss of Zeb1 in HSCs activated transcriptional programs of deregulated HSC maintenance and multilineage differentiation genes and of cell polarity consisting of cytoskeleton-, lipid metabolism/lipid membrane-, and cell adhesion-related genes. Notably, epithelial cell adhesion molecule (EpCAM) expression was prodigiously upregulated in Zeb1-KO HSCs, which correlated with enhanced cell survival, diminished mitochondrial metabolism, ribosome biogenesis, and differentiation capacity and an activated transcriptomic signature associated with acute myeloid leukemia (AML) signaling. ZEB1 expression was downregulated in AML patients, and Zeb1 KO in the malignant counterparts of HSCs - leukemic stem cells (LSCs) - accelerated MLL-AF9- and Meis1a/Hoxa9-driven AML progression, implicating Zeb1 as a tumor suppressor in AML LSCs. Thus, Zeb1 acts as a transcriptional regulator in hematopoiesis, critically coordinating HSC self-renewal, apoptotic, and multilineage differentiation fates required to suppress leukemic potential in AML.

Gata2 as a Crucial Regulator of Stem Cells in Adult Hematopoiesis and Acute Myeloid Leukemia.

Subversion of transcription factor (TF) activity in hematopoietic stem/progenitor cells (HSPCs) leads to the development of therapy-resistant leukemic stem cells (LSCs) that drive fulminant acute myeloid leukemia (AML). Using a conditional mouse model where zinc-finger TF Gata2 was deleted specifically in hematopoietic cells, we show that knockout of Gata2 leads to rapid and complete cell-autonomous loss of adult hematopoietic stem cells. By using short hairpin RNAi to target GATA2, we also identify a requirement for GATA2 in human HSPCs. In Meis1a/Hoxa9-driven AML, deletion of Gata2 impedes maintenance and self-renewal of LSCs. Ablation of Gata2 enforces an LSC-specific program of enhanced apoptosis, exemplified by attenuation of anti-apoptotic factor BCL2, and re-instigation of myeloid differentiation--which is characteristically blocked in AML. Thus, GATA2 acts as a critical regulator of normal and leukemic stem cells and mediates transcriptional networks that may be exploited therapeutically to target key facets of LSC behavior in AML.

Inhibition of GATA2 restrains cell proliferation and enhances apoptosis and chemotherapy mediated apoptosis in human GATA2 overexpressing AML cells.

GATA2, a zinc finger transcription factor predominantly expressed in hematopoietic cells, acts as an essential regulator of hematopoietic stem cell generation, survival and functionality. Loss and gain of GATA2 expression has been implicated in myelodysplastic syndrome and acute myeloid leukemia (AML) yet the precise biological impact of GATA2 expression on human AML cell fate decisions remains ambiguous. Herein, we performed large-scale bioinformatics that demonstrated relatively frequent GATA2 overexpression in AML patients as well as select human AML (or AML-like) cell lines. By using shRNAi to target GATA2 in these AML cell lines, and an AML cell line expressing normal levels of GATA2, we found that inhibition of GATA2 caused attenuated cell proliferation and enhanced apoptosis exclusively in AML cell lines that overexpress GATA2. We proceeded to pharmacologically inhibit GATA2 in concert with AML chemotherapeutics and found this augmented cell killing in AML cell lines that overexpress GATA2, but not in an AML cell line expressing normal levels of GATA2. These data indicate that inhibition of GATA2 enhances chemotherapy-mediated apoptosis in human AML cells overexpressing GATA2. Thus, we define novel insights into the oncogenic role of GATA2 in human AML cells and suggest the potential utilization of transient GATA2 therapeutic targeting in AML.

Isolation and Characterisation of Human Adipose-Derived Stem Cells.

Recently, adipose-derived stem cells (ASCs), obtained from fresh human lipoaspirate, have shown promise as immunomodulatory agents having demonstrated immunosuppressive functionality both in vitro and in vivo. A number of researchers have described the isolation of ASCs through the enzymatic digestion of fat samples, followed by a number of purification steps, involving centrifugation and filtration. Here, we utilize a standard isolation technique, with the added purification of putative ASCs by fluorescence activated cell sorting (FACS). ASCs are an extremely valuable resource in clinical applications, including reconstruction, regeneration, and investigations into immune activity. This method could be used to isolate and purify ASCs for such downstream applications.

Stem Cells Cycle toward Immune Surveillance.

Immune surveillance is an established regulatory mechanism that spares tissues from malignant transformation. Agudo et al. (2018) find that the chief cell type to generate tissues in the body-somatic stem cells-is subject to immune surveillance only during proliferation.

Dickkopf-3, a tissue-derived modulator of local T-cell responses.

The adaptive immune system protects organisms from harmful environmental insults. In parallel, regulatory mechanisms control immune responses in order to assure preservation of organ integrity. Yet, molecules involved in the control of T-cell responses in peripheral tissues are poorly characterized. Here, we investigated the function of Dickkopf-3 in the modulation of local T-cell reactivity. Dkk3 is a secreted, mainly tissue-derived protein with highest expression in organs considered as immune-privileged such as the eye, embryo, placenta, and brain. While T-cell development and activation status in naïve Dkk3-deficient mice was comparable to littermate controls, we found that Dkk3 contributes to the immunosuppressive microenvironment that protects transplanted, class-I mismatched embryoid bodies from T-cell-mediated rejection. Moreover, genetic deletion or antibody-mediated neutralization of Dkk3 led to an exacerbated experimental autoimmune encephalomyelitis (EAE). This phenotype was accompanied by a change of T-cell polarization displayed by an increase of IFNγ-producing T cells within the central nervous system. In the wild-type situation, Dkk3 expression in the brain was up-regulated during the course of EAE in an IFNγ-dependent manner. In turn, Dkk3 decreased IFNγ activity and served as part of a negative feedback mechanism. Thus, our findings suggest that Dkk3 functions as a tissue-derived modulator of local CD4(+) and CD8(+) T-cell responses.

Lack of immune response to differentiated cells derived from syngeneic induced pluripotent stem cells.

The prospects for using autologous induced pluripotent stem cells (iPSCs) in cell replacement therapy have been tempered by evidence that undifferentiated, syngeneic mouse iPSCs are immunogenic upon transplantation. However, the immunogenicity of more therapeutically relevant differentiated cells remains unexplored. Here, we differentiated mouse iPSCs into embryoid bodies (EBs) or representative cell types spanning the three embryonic germ layers and assessed their immunogenicity in vitro and after their transplantation into syngeneic recipients. We found no evidence of increased T cell proliferation in vitro, rejection of syngeneic iPSC-derived EBs/tissue-specific cells (TSCs) after transplantation, or an antigen-specific secondary immune response. Thus, differentiated cells derived from syngeneic iPSCs do not appear to be rejected after transplantation. We also found little evidence of an immune response to undifferentiated, syngeneic iPSCs. Our data support the idea that differentiated cells generated from autologous iPSCs could be applied for cell replacement therapy without eliciting immune rejection.

Concise review: Immune recognition of induced pluripotent stem cells.

Autologous-induced pluripotent stem cells (iPSCs) may eventually be used in cell replacement therapies to treat a wide range of diseases and have been touted as a solution to the vexing problem of immune rejection in this context. Emerging evidence suggests, however, that ostensibly histocompatible iPSCs may be rejected following transplantation. Here, we review the mechanisms that contribute to immunogenicity in iPSCs and forward approaches to permit their acceptance in potential cell replacement therapies.

A role for regulatory T cells in acceptance of ESC-derived tissues transplanted across an major histocompatibility complex barrier.

We have previously reported that ESC-derived tissues are subject to some level of immune privilege, which might facilitate induction of immune tolerance. Herein, we further demonstrate that fully allogeneic ESC-derived tissues are accepted with a regimen of coreceptor blockade even in recipients known to be relatively resistant to such a tolerizing protocol. Moreover, ESC-derived tissues could be spontaneously accepted across a class I major histocompatibility complex disparity. We further show that CD4(+)FoxP3(+) regulatory T cells (Treg) appear to be essential for this natural "privileged" state as their ablation with an anti-CD25 mAb results in rejection of ESC-derived tissue. This same treatment exposes activation of macrophages and effector CD8(+) T cells, suggesting that these cells are subject to regulatory T cell control. Thus, spontaneous acceptance of ESC-derived tissues mimics the acquired immune privilege induced by coreceptor blockade and is determined by Treg-mediated suppression.

Characteristics of the early immune response following transplantation of mouse ES cell derived insulin-producing cell clusters.

BACKGROUND: The fully differentiated progeny of ES cells (ESC) may eventually be used for cell replacement therapy (CRT). However, elements of the innate immune system may contribute to damage or destruction of these tissues when transplanted. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we assessed the hitherto ill-defined contribution of the early innate immune response in CRT after transplantation of either ESC derived insulin producing cell clusters (IPCCs) or adult pancreatic islets. Ingress of neutrophil or macrophage cells was noted immediately at the site of IPCC transplantation, but this infiltration was attenuated by day three. Gene profiling identified specific inflammatory cytokines and chemokines that were either absent or sharply reduced by three days after IPCC transplantation. Thus, IPCC transplantation provoked less of an early immune response than pancreatic islet transplantation. CONCLUSIONS/SIGNIFICANCE: Our study offers insights into the characteristics of the immune response of an ESC derived tissue in the incipient stages following transplantation and suggests potential strategies to inhibit cell damage to ensure their long-term perpetuation and functionality in CRT.

Approaches for immunological tolerance induction to stem cell-derived cell replacement therapies.

The shortage of donors for organ transplantation and also to treat degenerative diseases has led to the development of the new field of regenerative medicine. One aim of this field, in addition to in vivo induction of endogenous tissue regeneration, is to utilize stem cells as a supplementary source of cells to repair or replace tissues or organs that have ceased to function owing to ageing or autoimmunity. Embryonic stem cells hold promise in this respect because of their developmental capacity to generate all tissues within the body. More recently, the discovery of induced pluripotent stem cells, somatic cells reprogrammed to a primitive embryonic-like state by the introduction of pluripotency factors, may also act as an important cell source for cell replacement therapy. However, before cell replacement therapy can become a reality, one must consider how to overcome the potential transplant rejection of stem cell-derived products. There are several potential ways to circumvent the hurdles presented by the immune system in this setting, not least the induction of immunological tolerance in the host. In this review, we consider this and other approaches for engendering acceptance of stem cell-derived tissues.

Variation in MHC expression between undifferentiated mouse ES cells and ES cell-derived insulin-producing cell clusters.

BACKGROUND: The progeny of embryonic stem (ES) cells may eventually be used to replace damaged tissues in transplantation, yet their immunogenicity remains ill-defined. The major histocompatibility complex (MHC) is a determinant of immunogenicity in transplantation. METHODS AND RESULTS: Herein, we show differences in MHC expression between mouse ES cells and ES cell derived insulin producing cell clusters (IPCCs), including a relatively higher expression of MHC Class I in IPCCs and a faster, more dramatic induction of MHC Class I in IPCCs following challenge with interferon-gamma (IFN-gamma). MHC Class II was induced on IPCCs, but not ES cells, after exposure to IFN-gamma. Transplantation of syngeneic or allogeneic IPCCs was insufficient to trigger up-regulation of MHC class I within three days after transplantation. DISCUSSION: These data highlight differences in MHC expression between ES cells and a fully differentiated ES cell derived tissue and suggest how the progeny of ES cells may be susceptible to rejection after transplantation.

GATA-2 regulates granulocyte-macrophage progenitor cell function.

The zinc finger transcription factor GATA-2 has been implicated in the regulation of hematopoietic stem cells. Herein, we explored the role of GATA-2 as a candidate regulator of the hematopoietic progenitor cell compartment. We showed that bone marrow from GATA-2 heterozygote (GATA-2(+/-)) mice displayed attenuated granulocyte-macrophage progenitor function in colony-forming cell (CFC) and serial replating CFC assays. This defect was mapped to the Lin(-)CD117(+)Sca-1(-)CD34(+)CD16/32(high) granulocyte-macrophage progenitor (GMP) compartment of GATA-2(+/-) marrow, which was reduced in size and functionally impaired in CFC assays and competitive transplantation. Similar functional impairments were obtained using a RNA interference approach to stably knockdown GATA-2 in wild-type GMP. Although apoptosis and cell-cycle distribution remained unperturbed in GATA-2(+/-) GMP, quiescent cells from GATA-2(+/-) GMP exhibited altered functionality. Gene expression analysis showed attenuated expression of HES-1 mRNA in GATA-2-deficient GMP. Binding of GATA-2 to the HES-1 locus was detected in the myeloid progenitor cell line 32Dcl3, and enforced expression of HES-1 expression in GATA-2(+/-) GMP rectified the functional defect, suggesting that GATA-2 regulates myeloid progenitor function through HES-1. These data collectively point to GATA-2 as a novel, pivotal determinant of GMP cell fate.

A comparison of protocols used to generate insulin-producing cell clusters from mouse embryonic stem cells.

Embryonic stem cells (ESCs) have the capacity to generate a panoply of tissue types and may therefore provide an alternative source of tissue in regenerative medicine to treat potentially debilitating conditions like Type 1 diabetes mellitus. However, the ability of mouse ESCs to generate insulin-producing cell clusters (IPCCs) remains highly contentious. In an attempt to clarify this issue, three protocols for the ESC-based generation of IPCCs (referred to as Blyszczuk, Hori, and Lumelsky protocols) were modified and evaluated for their ability to express pancreatic islet genes and proteins and their capacity to function. Herein, we show that the Blyszczuk protocol reproducibly generated IPCCs with gene-expression characteristics that were qualitatively and quantitatively most reminiscent of those found in pancreatic islets. Furthermore, compared to the Hori and Lumelsky protocols, Blyszczuk-derived IPCCs exhibited superior expression of c-peptide, a by-product of de novo insulin synthesis. Functionally, Blyszczuk IPCCs, in contrast to Hori and Lumelsky IPCCs, were able to transiently restore normal blood glucose levels in diabetic mice (<1 week). Longer normoglycemic rescue (>2 weeks) was also achieved in a third of diabetic recipients receiving Blyszczuk IPCCs. Yet Blyszczuk IPCCs were less able to rescue experimental diabetes than isolated syngeneic pancreatic islet tissue. Therefore, depending on the mode of differentiation, ESCs can be driven to generate de novo IPCCs that possess limited functionality. Further modifications to differentiation protocols will be essential to improve the generation of functional IPCCs from mouse ESCs.

Embryonic stem cell transplantation: potential applicability in cell replacement therapy and regenerative medicine.

Embryonic stem cells are derived from the inner cell mass of the trophoblast, and have the ability to differentiate into all the tissues of the fetus. As such, their potential in cell replacement therapy and regenerative medicine has been widely acknowledged. Useful cell types such as neurons, cardiomyocytes, hepatocytes, pancreatic beta cells, and blood cells have all been successfully derived in the laboratory. Furthermore, embryonic stem cells may be utilized in novel immunomodulatory applications, such as hematopoietic chimerism strategies aimed at inducing tolerance to donor organ allografts. Unfortunately, progress in embryonic stem cell therapeutics continues to be hindered by haphazard differentiation and tumorigenesis; and the immune response to an embryonic stem cell-derived tissue graft is still an open question. This review summarizes the current state of embryonic stem cell research in regards to transplantation, highlighting the successes to date and the future obstacles yet to be overcome. Although embryonic stem cells are still far from their debut in the clinic, continued scientific advances engender optimism that they will eventually play an important role in cell replacement therapy and regenerative medicine.

Transplanting stem cells: potential targets for immune attack. Modulating the immune response against embryonic stem cell transplantation.

The curative promise of stem cells and their descendants for tissue regeneration and repair is currently the subject of an intense research effort worldwide. If it proves feasible to differentiate stem cells into specific tissues reliably and safely, this approach will be invaluable in the treatment of diseases that lead to organ degeneration or failure, providing an alternative or supplementary source of tissue for transplantation. Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of a pre-implantation blastocyst that can produce all cells and tissues of the foetus. In recent years, several laboratories have described the directed differentiation of ES cells into multiple mature cell types including: cardiomyocytes; haemopoietic cells; hepatocytes; neurones; muscle cells and both endocrine and exocrine cells of the pancreas. How the immune system of the host will respond when these ES cell-derived mature cells are transplanted is ill defined. This review will focus on the potential mechanisms that the immune system could use to target ES cell-derived transplants and how unwanted responses might be prevented.