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The most common cause of untreatable vision loss is dysfunction of the retina. Conditions, such as age-related macular degeneration, diabetic retinopathy and glaucoma remain leading causes of untreatable blindness worldwide. Various stem cell approaches are being explored for treatment of retinal regeneration. The rationale for using bone marrow stem cells to treat retinal dysfunction is based on preclinical evidence showing that bone marrow stem cells can rescue degenerating and ischemic retina. These stem cells have primarily paracrine trophic effects although some cells can directly incorporate into damaged tissue. Since the paracrine trophic effects can have regenerative effects on multiple cells in the retina, the use of this cell therapy is not limited to a particular retinal condition. Autologous bone marrow-derived stem cells are being explored in early clinical trials as therapy for various retinal conditions. These bone marrow stem cells include mesenchymal stem cells, mononuclear cells and CD34 + cells. Autologous therapy requires no systemic immunosuppression or donor matching. Intravitreal delivery of CD34 + cells and mononuclear cells appears to be tolerated and is being explored since some of these cells can home into the damaged retina after intravitreal administration. The safety of intravitreal delivery of mesenchymal stem cells has not been well established. This review provides an update of the current evidence in support of the use of bone marrow stem cells as treatment for retinal dysfunction. The potential limitations and complications of using certain forms of bone marrow stem cells as therapy are discussed. Future directions of research include methods to optimize the therapeutic potential of these stem cells, non-cellular alternatives using extracellular vesicles, and in vivo high-resolution retinal imaging to detect cellular changes in the retina following cell therapy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mesenchymal stem cells (MSCs) are an excellent source for numerous cellular therapies due to their simple isolation, low immunogenicity, multipotent differentiation potential and regenerative secretion profile. However, over-expanded MSCs show decreased therapeutic efficacy. This shortcoming may be circumvented by identifying methods that promote self-renewal of MSCs in culture. HMGA2 is a DNA-binding protein that regulates self-renewal in multiple types of stem cells through chromatin remodeling, but its impact on human bone marrow-derived MSCs is not known. Using an isolation method to obtain pure MSCs within 9 days in culture, we show that expression of HMGA2 quickly decreases during early expansion of MSCs, while let-7 microRNAs (which repress HMGA2) are simultaneously increased. Remarkably, we demonstrate that FGF-2, a growth factor commonly used to promote self-renewal in MSCs, rapidly induces HMGA2 expression in a time- and concentration-dependent manner. The signaling pathway involves FGF-2 receptor 1 (FGFR1) and ERK1/2, but acts independent from let-7. By silencing HMGA2 using shRNAs, we demonstrate that HMGA2 is necessary for MSC proliferation. However, we also show that over-expression of HMGA2 does not increase cell proliferation, but rather abrogates the mitogenic effect of FGF-2, possibly through inhibition of FGFR1. In addition, using different methods to assess in vitro differentiation, we show that modulation of HMGA2 inhibits adipogenesis, but does not affect osteogenesis of MSCs. Altogether, our results show that HMGA2 expression is associated with highly proliferating MSCs, is tightly regulated by FGF-2, and is involved in both proliferation and adipogenesis of MSCs. J. Cell. Biochem. 117: 2128-2137, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Purpose: Intravitreal murine lineage-negative bone marrow (BM) hematopoietic cells slow down retinal degeneration. Because human BM CD34+ hematopoietic cells are not precisely comparable to murine cells, this study examined the effect of intravitreal human BM CD34+ cells on the degenerating retina using a murine model., Methods: C3H/HeJrd1/rd1 mice, immunosuppressed systemically with tacrolimus and rapamycin, were injected intravitreally with PBS (n = 16) or CD34+ cells (n = 16) isolated from human BM using a magnetic cell sorter and labeled with enhanced green fluorescent protein (EGFP). After 1 and 4 weeks, the injected eyes were imaged with scanning laser ophthalmoscopy (SLO)/optical coherence tomography (OCT) and tested with electroretinography (ERG). Eyes were harvested after euthanasia for immunohistochemical and microarray analysis of the retina., Results: In vivo SLO fundus imaging visualized EGFP-labeled cells within the eyes following intravitreal injection. Simultaneous OCT analysis localized the EGFP-labeled cells on the retinal surface resulting in a saw-toothed appearance. Immunohistochemical analysis of the retina identified EGFP-labeled cells on the retinal surface and adjacent to ganglion cells. Electroretinography testing showed a flat signal both at 1 and 4 weeks following injection in all eyes. Microarray analysis of the retina following cell injection showed altered expression of more than 300 mouse genes, predominantly those regulating photoreceptor function and maintenance and apoptosis., Conclusions: Intravitreal human BM CD34+ cells rapidly home to the degenerating retinal surface. Although a functional benefit of this cell therapy was not seen on ERG in this rapidly progressive retinal degeneration model, molecular changes in the retina associated with CD34+ cell therapy suggest potential trophic regenerative effects that warrant further exploration.

Purpose: To determine long-term safety of intravitreal administration of good manufacturing practice (GMP)-grade human bone-marrow-derived CD34(+) cells in NOD-SCID (nonobese diabetic-severe combined immunodeficiency) mice with acute retinal ischemia-reperfusion injury, a model for retinal vasculopathy., Method: Acute ischemia-reperfusion injury was induced in the right eye of adult NOD-SCID mice (n = 23) by transient elevation of intraocular pressure. Seven days later, 12 injured eyes and 5 normal contralateral eyes were injected each intravitreally with 5 × 10(4) CD34(+) cells isolated under GMP conditions from a healthy human donor bone marrow using an immunomagnetic cell isolation system. The remaining 11 injured eyes were not treated and served as controls. Mice were euthanized 1 day, 4 months, and 8 months later. Both eyes were enucleated and examined by immunohistochemical analysis and hematoxylin and eosin staining. Among mice followed for 8 months, electroretinography (ERG) was performed on both eyes before euthanization. All major organs were examined grossly and histologically after serial sectioning., Results: Immunohistochemical staining 4 months after injection showed detectable CD34(+) cells in the retinal vasculature. ERG at 8 months after CD34(+) cell injection showed signals that were similar in untreated eyes. Histology of the enucleated eyes injected with CD34(+) cells showed no intraocular tumor or abnormal tissue growth after 8 months. Histologic analysis of all major organs showed no abnormal proliferation of human cells., Conclusions: Intravitreal administration of GMP-grade human bone-marrow-derived CD34(+) cells appears to be well tolerated long-term in eyes with acute retinal ischemic injury. A clinical trial will start to further explore this therapy.

A key mechanism for mesenchymal stem cells/bone marrow stromal cells (MSCs) to promote tissue repair is by secretion of soluble growth factors (GFs). Therefore, clinical application could be optimized by a combination of cell and gene therapies, where MSCs are genetically modified to express higher levels of a specific factor. However, it remains unknown how this overexpression may alter the fate of the MSCs. Here, we show effects of overexpressing the growth factors, such as basic fibroblast growth factor (bFGF), platelet derived growth factor B (PDGF-BB), transforming growth factor β(1) (TGF-β(1) ), and vascular endothelial growth factor (VEGF), in human bone marrow-derived MSCs. Ectopic expression of bFGF or PDGF-B lead to highly proliferating MSCs and lead to a robust increase in osteogenesis. In contrast, adipogenesis was strongly inhibited in MSCs overexpressing PDGF-B and only mildly affected in MSCs overexpressing bFGF. Overexpression of TGF-β(1) blocked both osteogenic and adipogenic differentiation while inducing the formation of stress fibers and increasing the expression of the smooth muscle marker calponin-1 and the chondrogenic marker collagen type II. In contrast, MSCs overexpressing VEGF did not vary from control MSCs in any parameters, likely due to the lack of VEGF receptor expression on MSCs. MSCs engineered to overexpress VEGF strongly induced the migration of endothelial cells and enhanced blood flow restoration in a xenograft model of hind limb ischemia. These data support the rationale for genetically modifying MSCs to enhance their therapeutically relevant trophic signals, when safety and efficacy can be demonstrated, and when it can be shown that there are no unwanted effects on their proliferation and differentiation., (Copyright © 2011 AlphaMed Press.)

Within the bone marrow (BM), hematopoietic progenitor cells (HPCs) are localized in poorly oxygenated niches where they interact with the surrounding osteoblasts (OBs) through VLA4/VCAM-1 engagement, and are exposed to interleukin-6 (IL-6), stem cell factor (SCF), and chemokines such as CXCL12 (OB factors). Umbilical cord (UC) is more highly oxygenated that the BM microenvironment. When UC-HPCs are exposed to the 2% to 3% O(2) concentration found in the bone endosteum, their survival is significantly decreased. However, engagement of VLA-4 integrins on UCB-derived CD34(+) cells reduced cell death in 2% to 3% O(2) conditions, which was associated with an increase in phospho-Ser473 AKT and an increase in phospho-Ser9 GSK3b. Consistent with the role of GSK3b in destabilizing beta-catenin, there was more cytoplasmic beta-catenin in UC-HPCs exposed to 2% to 3% O(2) on fibronectin, compared with suspension culture. UC-HPCs cultured at 2% to 3% O(2) with OB factors showed an increase in nuclear beta-catenin and persistence of a small pool of CD34(+)38(-) HPCs. CFU assays followed by surface phenotyping of the plated colonies showed improved maintenance of mixed lineage colonies with both erythroid and megakaryocytic precursors. These studies provide a biologic perspective for how UC-derived HPCs adapt to the bone endosteum, which is low in oxygen and densely populated by osteoblasts.

The bone is the third most common site of cancer metastasis. To invade the bone, tumor cells produce osteoclast-activating factors that increase bone resorption by osteoclasts. Here we report that human neuroblastoma cells that form osteolytic lesions in vivo do not produce osteoclast-activating factors but rather stimulate osteoclast activity in the presence of human bone marrow mesenchymal stem cells. This alternative pathway of osteoclast activation involves a nonadhesive interaction between neuroblastoma cells and bone marrow mesenchymal stem cells. Stimulated bone marrow mesenchymal stem cells express markedly increased levels of interleukin-6, which is then responsible for osteoclast activation. This report describes a critical role of bone marrow mesenchymal stem cells in bone destruction in cancer.

Duchenne muscular dystrophy (DMD) is a severe progressive muscle-wasting disorder caused by mutations in the dystrophin gene. Studies have shown that bone marrow cells transplanted into lethally irradiated mdx mice, the mouse model of DMD, can become part of skeletal muscle myofibers. Whether human marrow cells also have this ability is unknown. Here we report the analysis of muscle biopsies from a DMD patient (DMD-BMT1) who received bone marrow transplantation at age 1 year for X-linked severe combined immune deficiency and who was diagnosed with DMD at age 12 years. Analysis of muscle biopsies from DMD-BMT1 revealed the presence of donor nuclei within a small number of muscle myofibers (0.5-0.9%). The majority of the myofibers produce a truncated, in-frame isoform of dystrophin lacking exons 44 and 45 (not wild-type). The presence of bone marrow-derived donor nuclei in the muscle of this patient documents the ability of exogenous human bone marrow cells to fuse into skeletal muscle and persist up to 13 years after transplantation.

We have previously described extrathymic generation of human T cells from purified stem cells in the bone marrow of athymic immune deficient mice. This system provides a pure population of extrathymic human T cells that is devoid of contamination by peripheral expansion of thymic-selected T cells. In the current studies, we phenotypically compared the extrathymic human T cells (Ex-T) to T cells from human peripheral blood leukocytes (PBL), umbilical cord blood (CB), bone marrow (BM), and postnatal thymus. There were few CD4(+)/CD8(+) double positive (DP) cells in PBL, CB, BM, and Ex-T, in comparison with over 85% DP cells in thymus. More CD8(+) and CD4(dim) cells were observed in Ex-T than in the thymic-selected cells. Ex-T and T cells in thymus and peripheral tissues differed in their CD8 isoforms. There were more TCRgamma/delta T cells in PBL, CB, BM, and Ex-T than in thymus. Similar to the bright CD3(+) T cells in thymus, T cells in PBL, CB, and BM were CD3 bright and expressed the adhesion molecules CD44 and L-selectin (CD62L), while intermediate CD3 T cells in thymus lacked CD44 and L-selectin. However, the majority of Ex-T only expressed CD44 but not L-selectin. In summary, thymic- and extrathymic-derived T cells are phenotypically different. The identification of extrathymically derived T cells in humans will allow us to begin to understand their role in the early contribution to immune recovery posttransplantation and their possible involvement in autoimmunity and other disease states., (Copyright 2001 Academic Press.)

The beige/nude/xid/human (bnx/hu) model of human hematopoiesis provides a unique opportunity to study extrathymic human T lymphocyte development in an in vivo system. Purified human hematopoietic stem cells develop into mature T lymphocytes and immature progenitors in the bone marrow of athymic bnx mice. The human T cells are all TCR alpha beta(+) and display a restricted TCRV beta repertoire. In the current studies, we examined the effects of systemic human IL-7 (huIL-7) administration on the phenotype and the activation status of the bnx/hu T cells. In the majority of the mice that did not have huIL-7 administration, a higher frequency of human CD3(+)/CD8(+) than CD3(+)/CD4(+) T cells developed in the bone marrow. This phenomenon is also frequently observed in human bone marrow transplant recipients. Extremely low levels of IL-2 were expressed by human CD3(+) cells isolated from these mice, in response to PMA plus ionomycin and to CD3 and CD28 cross-linking. IL-4 was not expressed by cells exposed to either stimulus, demonstrating a profound inability of the bnx/hu T cells to produce this cytokine. Systemic production of huIL-7 from engineered stromal cells transplanted into the mice increased the human CD4 to CD8 ratios, and increased the ratio of memory to naive CD4(+) and CD8(+) T cells. The human CD3(+) cells recovered from mice that had systemic huIL-7 and equivalent numbers of CD3(+)/CD4(+) and CD3(+)/CD8(+) cells in the marrow were still unable to produce IL-4 in response to any condition tested, but were capable of normal levels of IL-2 production following stimulation.

Retroviral gene transfer of the glucocerebrosidase gene to hematopoietic progenitor and stem cells has shown promising results in animal models and corrected the enzyme deficiency in cells from Gaucher patients in vitro. Therefore, a clinical protocol was initiated to explore the safety and feasibility of retroviral transduction of peripheral blood (PB) or bone marrow (BM) CD34+ cells with the G1Gc vector. This vector uses the viral LTR promoter to express the human glucocerebrosidase cDNA. Three adult patients have been entered with follow-up of 6-15 months. Target cells were G-CSF-mobilized and CD34-enriched PB cells or CD34-enriched steady state BM cells, and were transduced ex vivo for 72 hr. Patient 1 had PB cells transduced in the presence of autologous stromal marrow cells. Patient 2 had PB cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. Patient 3 had BM cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. At the end of transduction, the cells were collected and infused immediately without any preparative treatment of the patients. The transduction efficiency of the CD34+ cells at the end of transduction was approximately 1, 10, and 1 for patients 1, 2, and 3, respectively, as estimated by semiquantitative PCR on bulk samples and PCR analysis of individual hematopoietic colonies. Gene marking in vivo was demonstrated in patients 2 and 3. Patient 2 had vector-positive PB granulocytes and mononuclear bone marrow cells at 1 month postinfusion and positive PB mononuclear cells at 2 and 3 months postinfusion. Patient 3 had a positive BM sample at 1 month postinfusion but was negative thereafter. These results indicate that gene-marked cells can engraft and persist for at least 3 months postinfusion, even without myeloablation. However, the level of corrected cells (<0.02%) is too low to result in any clinical benefit, and glucocerebrosidase enzyme activity did not increase in any patient following infusion of transduced cells. Modifications of vector systems and transduction conditions, along with partial myeloablation to allow higher levels of engraftment, may be necessary to achieve beneficial levels of correction in patients with Gaucher disease.

Human hematopoiesis can be supported in beige/nude/ XID (bnx) mice by coinjection of human bone marrow stromal cells engineered to secrete human interleukin 3 (HuIL-3). The major limitation is a total absence of human B cell development in the mice, which could be due to supraphysiological levels of HuIL-3 in the circulation. In an effort to obtain human B lymphoid, as well as T lymphoid and myeloid cell development in the mice, CD34+ cells were coinjected with human marrow stromal cells engineered to secrete human IL-2, IL-7, stem cell factor or FLT3 ligand, +/- IL-3. No single factor other than IL-3 supported sustained human hematopoiesis in the mice, although cytokines were expressed for four to six months post-transplantation. Production of both HuIL-3 and IL-7 in the mice supported extrathymic development of human T lymphocytes, but no B cells, myeloid cells, or clonogenic progenitors were detected. Human B cells were not produced from CD34+ cells in the bnx mice under any condition tested. Another limitation to the bnx/Hu system is a lack of maturation of human red blood cells, although BFU-E are maintained. Stromal cells secreting human erythropoietin and IL-3 were cotransplanted into mice with HuCD34+ cells and an increase in hematocrit from 40%-45% to 80%-85% resulted, with production of human and murine red blood cells. Unfortunately, all mice (n = 9) suffered strokes, displayed paralysis and died within three weeks. The bnx/Hu cotransplantation model provides an interesting system in which to study human hematopoietic cell differentiation under the influence of various cytokines.