Reeva Aggarwal

Hematopoietic Stem Cells: Transcriptional Regulation, Ex Vivo Expansion and Clinical Application

Maintenance of ex vivo hematopoietic stem cells (HSC) pool and its differentiated progeny is regulated by complex network of transcriptional factors, cell cycle proteins, extracellular matrix, and their microenvironment through an orchestrated fashion. Strides have been made to understand the mechanisms regulating in vivo quiescence and proliferation of HSCs to develop strategies for ex vivo expansion. Ex vivo expansion of HSCs is important to procure sufficient number of stem cells and as easily available source for HSC transplants for patients suffering from hematological disorders and malignancies. Our lab has established a nanofiber-based ex vivo expansion strategy for HSCs, while preserving their stem cell characteristics. Ex vivo expanded cells were also found biologically functional in various disease models. However, the therapeutic potential of expanded stem cells at clinical level still needs to be verified. This review outlines transcriptional factors that regulate development of HSCs and their commitment, genes that regulate cell cycle status, studies that attempt to develop an effective and efficient protocol for ex vivo expansion of HSCs and application of HSC in various non-malignant and malignant disorders. Overall the goal of the current review is to deliver an understanding of factors that are critical in resolving the challenges that limit the expansion of HSCs in vivo and ex vivo.

Human Umbilical Cord Blood-Derived CD34+ Cells Reverse Osteoporosis in NOD/SCID Mice by Altering Osteoblastic and Osteoclastic Activities

Background Osteoporosis is a bone disorder associated with loss of bone mineral density and micro architecture. A balance of osteoblasts and osteoclasts activities maintains bone homeostasis. Increased bone loss due to increased osteoclast and decreased osteoblast activities is considered as an underlying cause of osteoporosis. Methods and Findings The cures for osteoporosis are limited, consequently the potential of CD34+ cell therapies is currently being considered. We developed a nanofiber-based expansion technology to obtain adequate numbers of CD34+ cells isolated from human umbilical cord blood, for therapeutic applications. Herein, we show that CD34+ cells could be differentiated into osteoblastic lineage, in vitro. Systemically delivered CD34+ cells home to the bone marrow and significantly improve bone deposition, bone mineral density and bone micro-architecture in osteoporotic mice. The elevated levels of osteocalcin, IL-10, GM-CSF, and decreased levels of MCP-1 in serum parallel the improvements in bone micro-architecture. Furthermore, CD34+ cells improved osteoblast activity and concurrently impaired osteoclast differentiation, maturation and functionality. Conclusions These findings demonstrate a novel approach utilizing nanofiber-expanded CD34+ cells as a therapeutic application for the treatment of osteoporosis.

A Novel Technology for Hematopoietic Stem Cell Expansion Using Combination of Nanofiber and Growth Factors

Hematopoietic stem cell transplantation has been applied as a standard procedure of treatment for hematological disorders like multiple myeloma and leukemia for several decades. Various sources of stem cells like bone marrow, peripheral blood and umbilical cord blood are used for the transplantation. Among these umbilical cord blood is currently preferred due to the primitiveness of the derived stem cells and minimal possibilities of graft versus host disease or development of graft induced tumors. One of the problems for these sources is the procurement of sufficient number of donor stem cells. Inadequate number of cells may lead to delayed recovery and decrease survivability of the patient. Thus to overcome the limitation of stem cell number, development of an ex-vivo expansion technology is critically important. The recent emerging technology using nanofiber in combination with growth factors has made a significant improvement to the field of regenerative medicine and a couple of patents have been filed. In this review, we will focus on factors regulating hematopoietic stem cell self-renewal and expansion emphasizing on nanofiber as a supporting matrix.

Impact of Diffusion Barriers to Small Cytotoxic Molecules on the Efficacy of Immunotherapy in Breast Cancer

Molecular-focused cancer therapies, e.g., molecularly targeted therapy and immunotherapy, so far demonstrate only limited efficacy in cancer patients. We hypothesize that underestimating the role of biophysical factors that impact the delivery of drugs or cytotoxic cells to the target sites (for associated preferential cytotoxicity or cell signaling modulation) may be responsible for the poor clinical outcome. Therefore, instead of focusing exclusively on the investigation of molecular mechanisms in cancer cells, convection-diffusion of cytotoxic molecules and migration of cancer-killing cells within tumor tissue should be taken into account to improve therapeutic effectiveness. To test this hypothesis, we have developed a mathematical model of the interstitial diffusion and uptake of small cytotoxic molecules secreted by T-cells, which is capable of predicting breast cancer growth inhibition as measured both in vitro and in vivo. Our analysis shows that diffusion barriers of cytotoxic molecules conspire with γδ T-cell scarcity in tissue to limit the inhibitory effects of γδ T-cells on cancer cells. This may increase the necessary ratios of γδ T-cells to cancer cells within tissue to unrealistic values for having an intended therapeutic effect, and decrease the effectiveness of the immunotherapeutic treatment.

Human Ovarian Tumor Cells Escape γδ T Cell Recognition Partly by Down Regulating Surface Expression of MICA and Limiting Cell Cycle Related Molecules

Background Mechanisms of human Vγ2Vδ2 T cell-mediated tumor immunity have yet to be fully elucidated. Methods and Findings At least some tumor cell recognition is mediated by NKG2D-MICA interactions. Herein, by using MTT assay and PI-BrdU co-staining and Western-blot, we show that these Vγ2Vδ2 T cells can limit the proliferation of ovarian tumor cells by down regulation of apoptosis and cell cycle related molecules in tumor cells. Cell-to-cell contact is critical. γδ T cell-resistant, but not susceptible ovarian tumor cells escape γδ T cell-mediated immune recognition by up-regulating pErk1/2, thereby decreasing surface MICA levels. Erk1/2 inhibitor pretreatment or incubation prevents this MICA decrease, while up-regulating key cell cycle related molecules such as CDK2, CDK4 and Cyclin D1, as well as apoptosis related molecules making resistant tumor cells now vulnerable to γδ T cell-mediated lysis. Conclusion These findings demonstrate novel effects of γδT cells on ovarian tumor cells.

Genetic modification of ex-vivo expanded stem cells for clinical application.

Stem cell therapy is currently considered as an important regime for repairing, replacing or enhancing the biological functions of the damaged tissues. Among adult stem cells, hematopoietic stem cells (HSCs) are commonly used for cure of hematological disorders. However, the number of HSCs obtained from sources like bone marrow, peripheral or umbilical cord blood is not sufficient for routine clinical application. Thus, ex-vivo expansion of HSCs becomes critically important. Ex-vivo culture and expansion of stem cells are challenging, as stem cells differentiate in culture rather than self-renew. Lack of clarity about the factors responsible for quiescence and differentiation of HSCs, investigators struggled to optimize conditions for ex vivo expansion. As we understand better, various strategies can be incorporated to mimic in vivo conditions for successful expansion of stem cells. However, characterization and biological functionality should also be tested for expanded stem cells prior to clinical application. To treat ischemia by enhancing therapeutic angiogenesis and neo-vascularization, the role of genetic modification of HSCs with pro-angiogenic factors is the focus of this review.

Human Vγ2Vδ2 T cells limit breast cancer growth by modulating cell survival-, apoptosis-related molecules and microenvironment in tumors.

Innate immune system has been known to play an important role in inhibiting the malignant transformation, tumor progression and invasion. However, the mechanistic basis remains ambiguous. Despite polyclonality of human γδ T cells, Vγ2Vδ2 T cell subset was shown to recognize and limit the growth of various tumors at various degrees. The differential recognition of the tumor cells by Vγ2Vδ2 T cells are yet to be defined. Our study reveals that γδ T cells limit in vitro growth of most breast tumor cells, such as SkBr7 (HER2+), MCF7 (ER+) and MDA‐MB‐231 (ER−) by inhibiting their survival and inducing apoptosis, except BrCa‐MZ01 (PR+) cells. To investigate detail mechanisms of antineoplastic effects, we found that cell death was associated with the surface expression levels of MICA/B and ICAM1. Molecular signaling analysis demonstrated that inhibition of cell growth by γδ T cells was associated with the lower expression levels of cell survival‐related molecules such as AKT, ERK and concomitant upregulation of apoptosis‐related molecules, such as PARP, cleaved caspase 3 and tumor suppressor genes PTEN and P53. However, opposite molecular signaling was observed in the resistant cell line after coculture with γδ T cells. In vivo, antineoplastic effects of γδ T cells were also documented, where tumor growth was inhibited due to the downregulation of survival signals, strong induction of apoptotic molecules, disruption of microvasculature and increased infiltration of tumor associated macrophages. These findings reveal that a complex molecular signaling is involved in γδ T cell‐mediated antineoplastic effects.

Nanofiber-expanded human umbilical cord blood–derived CD34+ cell therapy accelerates cutaneous wound closure in NOD/SCID mice

Nanofiber‐expanded human umbilical cord blood–derived CD34+ cell therapy has been shown to have potential applications for peripheral and myocardial ischaemic diseases. However, the efficacies of expanded CD34+ cell therapy for treating cutaneous wounds and its mechanisms of action have yet to be established. Using an excisional wound model in non‐obese diabetic/severe combined immune deficient mice, we show herein that CD34+ cells accelerate the wound‐healing process by enhancing collagen synthesis, and increasing fibroblast cell migration within the wound bed. Concomitantly, reduced levels of matrix metalloproteinase (MMPs) such as MMP1, MMP3, MMP9 and MMP13 were detected in the wound beds of animals treated with CD34+ cells compared with vehicle‐treated controls. CD34+ cells were found to mediate enhanced migration and proliferation of dermal fibroblast cells in vitro. Moreover, CD34+ cells secrete collagen in a serum‐deprived environment. In mechanistic studies, co‐culture of CD34+ cells with primary skin fibroblasts increased the expression of collagen1A1, a component of type 1 collagen, and decreased the expression of MMP1 in fibroblast cells in the presence of a proteasome inhibitor. Finally, CD34+ cell–mediated functions were transcriptionally regulated by the c‐Jun N‐terminal kinases pathway. Collectively, these data provide evidence of therapeutic efficacy and a novel mechanism of nanofiber‐expanded CD34+ cell–mediated accelerated wound healing.

Kruppel-like factor 2 (KLF2) regulates monocyte differentiation and functions in mBSA and IL-1β-induced arthritis.

Kruppel-like factor 2 (KLF2) plays an important role in the regulation of a variety of immune cells, including monocytes. We have previously shown that KLF2 inhibits proinflammatory activation of monocytes. However, the role of KLF2 in arthritis is yet to be investigated. In the current study, we show that recruitment of significantly greater numbers of inflammatory subset of CD11b(+)F4/80(+)Ly6C+ monocytes to the inflammatory sites in KLF2 hemizygous mice compared to the wild type littermate controls. In parallel, inflammatory mediators, MCP-1, Cox-2 and PAI-1 were significantly up-regulated in bone marrow-derived monocytes isolated from KLF2 hemizygous mice, in comparison to wild-type controls. Methylated-BSA and IL-1β-induced arthritis was more severe in KLF2 hemizygous mice as compared to the littermate wild type controls. Consistent with this observation, monocytes isolated from KLF2 hemizygous mice showed an increased number of cells matured and differentiated towards osteoclastic lineage, potentially contributing to the severity of cartilage and bone damage in induced arthritic mice. The severity of arthritis was associated with the higher expression of proteins such as HSP60, HSP90 and MMP13 and attenuated levels of pPTEN, p21, p38 and HSP25/27 molecules in bone marrow cells of arthritic KLF2 hemizygous mice compared to littermate wild type controls. The data provide new insights and evidences of KLF2-mediated transcriptional regulation of arthritis via modulation of monocyte differentiation and function.

Nanofiber-expanded human umbilical cord blood-derived CD34 + cell therapy accelerates murine cutaneous wound closure by attenuating pro-inflammatory factors and secreting IL-10

Nanofiber-expanded human umbilical cord blood-derived CD34+ cell therapy is under consideration for treating peripheral and cardiac ischemia. However, the therapeutic efficacy of nanofiber-expanded human umbilical cord blood-derived (NEHUCB) CD34+ cell therapy for wound healing and its mechanisms are yet to be established. Using an excision wound model in NOD/SCID mice, we show herein that NEHUCB-CD34+ cells home to the wound site and significantly accelerate the wound-healing process compared to vehicle-treated control. Histological analysis reveals that accelerated wound closure is associated with the re-epithelialization and increased angiogenesis. Additionally, NEHUCB-CD34+ cell-therapy decreases expression of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6 and NOS2A in the wound bed, and concomitantly increases expression of IL-10 compared to vehicle-treated control. These findings were recapitulated in vitro using primary dermal fibroblasts and NEHUCB-CD34+ cells. Moreover, NEHUCB-CD34+ cells attenuate NF-κB activation and nuclear translocation in dermal fibroblasts through enhanced secretion of IL-10, which is known to bind to NF-κB and suppress transcriptional activity. Collectively, these data provide novel mechanistic evidence of NEHUCB-CD34+ cell-mediated accelerated wound healing.