Leslie E. Silberstein

The elusive nature and function of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a diverse subset of multipotent precursors present in the stromal fraction of many adult tissues and have drawn intense interest from translational and basic investigators. MSCs have been operationally defined by their ability to differentiate into osteoblasts, adipocytes and chondrocytes after in vitro expansion. Nevertheless, their identity in vivo, heterogeneity, anatomical localization and functional roles in adult tissue homeostasis have remained enigmatic and are only just starting to be uncovered.

Cardiomyocyte proliferation contributes to heart growth in young humans

The human heart is believed to grow by enlargement but not proliferation of cardiomyocytes (heart muscle cells) during postnatal development. However, recent studies have shown that cardiomyocyte proliferation is a mechanism of cardiac growth and regeneration in animals. Combined with evidence for cardiomyocyte turnover in adult humans, this suggests that cardiomyocyte proliferation may play an unrecognized role during the period of developmental heart growth between birth and adolescence. We tested this hypothesis by examining the cellular growth mechanisms of the left ventricle on a set of healthy hearts from humans aged 0–59 y (n = 36). The percentages of cardiomyocytes in mitosis and cytokinesis were highest in infants, decreasing to low levels by 20 y. Although cardiomyocyte mitosis was detectable throughout life, cardiomyocyte cytokinesis was not evident after 20 y. Between the first year and 20 y of life, the number of cardiomyocytes in the left ventricle increased 3.4-fold, which was consistent with our predictions based on measured cardiomyocyte cell cycle activity. Our findings show that cardiomyocyte proliferation contributes to developmental heart growth in young humans. This suggests that children and adolescents may be able to regenerate myocardium, that abnormal cardiomyocyte proliferation may be involved in myocardial diseases that affect this population, and that these diseases might be treatable through stimulation of cardiomyocyte proliferation.

Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment

The existence of a haematopoietic stem cell niche as a spatially confined regulatory entity relies on the notion that haematopoietic stem and progenitor cells (HSPCs) are strategically positioned in unique bone marrow microenvironments with defined anatomical and functional features. Here, we employ a powerful imaging cytometry platform to perform a comprehensive quantitative analysis of HSPC distribution in bone marrow cavities of femoral bones. We find that HSPCs preferentially localize in endosteal zones, where most closely interact with sinusoidal and non-sinusoidal bone marrow microvessels, which form a distinctive circulatory system. In situ tissue analysis reveals that HSPCs exhibit a hypoxic profile, defined by strong retention of pimonidazole and expression of HIF- 1α, regardless of localization throughout the bone marrow, adjacency to vascular structures or cell-cycle status. These studies argue that the characteristic hypoxic state of HSPCs is not solely the result of a minimally oxygenated niche but may be partially regulated by cell-specific mechanisms.

Functional expression of CXCR4 (CD184) on small-cell lung cancer cells mediates migration, integrin activation, and adhesion to stromal cells

Small-cell lung cancer (SCLC) is an aggressive, rapidly metastasizing neoplasm. The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) is constitutively secreted by marrow stromal cells and plays a key role for homing of hematopoietic cells to the marrow. Here, we report that tumor cells from patients with SCLC express high levels of functional CXCR4 receptors for the chemokine CXCL12. Reverse transcriptase–polymerase chain reaction and flow cytometry demonstrated CXCR4 mRNA and CXCR4 surface expression in SCLC cell lines. Immunohistochemistry of primary tumor samples from SCLC patients revealed high expression of CXCR4. CXCL12 elicited CXCR4 receptor endocytosis, actin polymerization, and a robust activation of phospho-p44/42 mitogen-activated protein kinase in SCLC cells. Furthermore, CXCL12 induced SCLC cell invasion into extracellular matrix and firm adhesion to marrow stromal cells. Stromal cell adhesion of SCLC cells was significantly inhibited by the specific CXCR4 antagonist T140, pertussis toxin, antivascular cell adhesion molecule-1(VCAM-1) antibodies, and CS-1 peptide, demonstrating the importance of CXCR4 chemokine receptor activation and α4β1 integrin binding, respectively. In addition, CXCL12 enhanced the adhesion of SCLC cells to immobilized VCAM-1, demonstrating that CXCR4 chemokine receptors can induce integrin activation on SCLC cells. As SCLC has a high propensity for bone marrow involvement, our findings suggest that CXCR4 chemokine receptors and α4β1 integrins play a critical role in the interaction of SCLC cells with stromal cells in the tumor microenvironment.

Production of autoantibodies by human-human hybridomas.

Peripheral blood lymphocytes and splenocytes of patients with autoimmune disease were used to prepare human-human hybridomas that produce autoantibodies. Because exogenous immunization was not used, the hybridoma antibodies were derived from B cells that spontaneously produced autoantibodies. 108 hybrids grew from 4,254 wells (2.5%). Optimal conditions for obtaining hybridomas with the GM 4672 myeloma line included initial growth in 2-ml wells, the use of 44% polyethylene glycol, a mononuclear cell/GM 4672 cell ratio 5:1, and prior stimulation of the B lymphocytes with pokeweed mitogen. Hybridoma supernatants had activity against ssDNA, platelets, and erythrocytes. The results demonstrate the feasibility of producing human-human hybridomas from lymphocytes of patients with various autoimmune diseases.

A Modified γ-Retrovirus Vector for X-Linked Severe Combined Immunodeficiency

BACKGROUND In previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus-based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancer-mediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1. METHODS We enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc). RESULTS All patients received bone marrow-derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients. CONCLUSIONS This modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.).

In vivo generation of transplantable human hematopoietic cells from induced pluripotent stem cells

Lineage-restricted cells can be reprogrammed to a pluripotent state known as induced pluripotent stem (iPS) cells through overexpression of 4 transcription factors. iPS cells are similar to human embryonic stem (hES) cells and have the same ability to generate all the cells of the human body, including blood cells. However, this process is extremely inefficient and to date has been unsuccessful at differentiating iPS into hematopoietic stem cells (HSCs). We hypothesized that iPS cells, injected into NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ immunocompromised (NSG) mice could give rise to hematopoietic stem/progenitor cells (HSPCs) during teratoma formation. Here, we report a novel in vivo system in which human iPS cells differentiate within teratomas to derive functional myeloid and lymphoid cells. Similarly, HSPCs can be isolated from teratoma parenchyma and reconstitute a human immune system when transplanted into immunodeficient mice. Our data provide evidence that in vivo generation of patient customized cells is feasible, providing materials that could be useful for transplantation, human antibody generation, and drug screening applications.

Bmi-1 is a target gene for SALL4 in hematopoietic and leukemic cells

Bmi-1 and SALL4 are putative oncogenes that modulate stem cell pluripotency and play a role in leukemogenesis. Murine Sall4 also has been shown to play an essential role in maintaining the properties of ES cells and governing the fate of the primitive inner cell mass. Here, we demonstrate that transcription from the Bmi-1 promoter is strikingly activated by SALL4 in a dose-dependent manner by using a luciferase reporter gene assay. Both promoter deletion construct studies and ChIP from a myeloid stem cell line, 32D, demonstrate that SALL4 binds to a specific region of the Bmi-1 promoter. Deletion of one copy of Sall4 by gene targeting in mouse bone marrow significantly reduced Bmi-1 expression. Reducing SALL4 expression by siRNA in the HL-60 leukemia cell line also results in significant down-regulation of Bmi-1. Furthermore, Bmi-1 expression is up-regulated in transgenic mice that constitutively overexpress human SALL4, and the levels of Bmi-1 in these mice increase as they progress from normal to preleukemic (myelodysplastic syndrome) and leukemic (acute myeloid leukemia) stages. High levels of H3–K4 trimethylation and H3–K79 dimethylation were observed in the SALL4 binding region of the Bmi-1 promoter. These findings suggest a novel link between SALL4 and Bmi-1 in regulating self-renewal of normal and leukemic stem cells. An increase in histone H3–K4 and H3–K79 methylation within the Bmi-1 promoter provides an epigenetic mechanism for histone modifications in SALL4-mediated Bmi-1 gene deregulation.

Stem Cell Factor SALL4 Represses the Transcriptions of PTEN and SALL1 through an Epigenetic Repressor Complex

Background The embryonic stem cell (ESC) factor, SALL4, plays an essential role in both development and leukemogenesis. It is a unique gene that is involved in self-renewal in ESC and leukemic stem cell (LSC). Methodology/Principal Findings To understand the mechanism(s) of SALL4 function(s), we sought to identify SALL4-associated proteins by tandem mass spectrometry. Components of a transcription repressor Mi-2/Nucleosome Remodeling and Deacetylase (NuRD) complex were found in the SALL4-immunocomplexes with histone deacetylase (HDAC) activity in ESCs with endogenous SALL4 expression and 293T cells overexpressing SALL4. The SALL4-mediated transcriptional regulation was tested on two potential target genes: PTEN and SALL1. Both genes were confirmed as SALL4 downstream targets by chromatin-immunoprecipitation, and their expression levels, when tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR), were decreased in 293T cells overexpressing SALL4. Moreover, SALL4 binding sites at the promoter regions of PTEN and SALL1 were co-occupied by NuRD components, suggesting that SALL4 represses the transcriptions of PTEN and SALL1 through its interactions with the Mi-2/NuRD complex. The in vivo repressive effect(s) of SALL4 were evaluated in SALL4 transgenic mice, where decreased expressions of PTEN and SALL1 were associated with myeloid leukemia and cystic kidneys, respectively. Conclusions/Significance In summary, we are the first to demonstrate that stem cell protein SALL4 represses its target genes, PTEN and SALL1, through the epigenetic repressor Mi-2/NuRD complex. Our novel finding provides insight into the mechanism(s) of SALL4 functions in kidney development and leukemogenesis.

Reducing the Risk for Transfusion-transmitted Cytomegalovirus Infection

OBJECTIVE To define the groups of patients at risk for transfusion-transmitted cytomegalovirus infection and to define the methods to reduce this risk. DATA SOURCES English-language publications on transfusion medicine. STUDY SELECTION AND DATA EXTRACTION Studies were selected that described cytomegalovirus infection in transfusion-dependent patients. Special attention was paid to reports that included observations about the prevalence and clinical manifestations of cytomegalovirus infection and recommendations for the prevention of infection. DATA SYNTHESIS Some patients with impaired immune responses who have never been exposed to cytomegalovirus are at risk for transfusion-transmitted cytomegalovirus infection. This infection, which is associated with substantial morbidity and mortality, can be avoided by additional screening of blood donors or by special processing of components for transfusion. CONCLUSIONS Transfusion products that are unlikely to transmit cytomegalovirus infection can be prepared by filtration to remove leukocytes or can be obtained by selecting donors who are seronegative for antibodies to cytomegalovirus. These products are indicated for certain groups of immunosuppressed patients, including pregnant women who are cytomegalovirus seronegative, premature infants of low birth weight who are born to cytomegalovirus-seronegative mothers, cytomegalovirus-seronegative recipients of allogeneic bone marrow transplants from cytomegalovirus-seronegative donors, and cytomegalovirus-seronegative patients with the acquired immunodeficiency syndrome (AIDS).