Development Of Novel Cell-based Therapies Research Articles

Controlled hydrogel-based encapsulation of macrophages determines cell survival and functionality upon cryopreservation

The development of novel cell-based therapies has increased the necessity to improve the long-term storage of cells. The current method of cryopreservation is far from optimal, causing ice-associated mechanical and osmotic damage to sensitive cells. Cell encapsulation is emerging as a new strategy to overcome those current limitations; however, few data are applicable to slow freezing, with conflicting results and multiple experimental conditions. The objective of this research work was to evaluate the impact of capsule size and encapsulation method on cell survival and functionality after a conventional freezing protocol. To this end, cells were encapsulated in alginate beads of different sizes, spanning the range of 200–2000 µm thanks to multiple extrusion techniques and conditions, and further cryopreserved using a slow cooling rate (−1°C/min) and 10 % DMSO as cryoprotectant. Our data show that there is a strong correlation between bead size and cell survival after a slow cooling cryopreservation process, with cell viabilities ranging from 7 to 70 % depending on the capsule size, with the smallest capsules (230 µm) achieving the highest level of survival. The obtained results indicate that the beads’ diameter, rather than their morphology or the technique used, plays a significant role in the post-thawing cell survival and functionality. These results show that a fine control of cell encapsulation in alginate hydrogels is required when it comes to overcoming the current limitations of long-term preservation techniques by slow cooling.

Exosomal HMGB1 derived from hypoxia-conditioned bone marrow mesenchymal stem cells increases angiogenesis via the JNK/HIF-1α pathway.

Mesenchymal stem cells (MSCs) have been described to induce angiogenesis in various tissues and have been used for the development of novel cell‐based therapies. Increasing evidence suggests that MSCs execute their paracrine function via the secretion of exosomes, especially under hypoxic conditions. However, the mechanisms by which MSC‐derived exosomes secreted under hypoxia enhance angiogenesis still remain unclear. To study exosome physiology under hypoxic or normoxic conditions, we isolated exosomes from bone marrow mesenchymal stem cells (BMSCs). Furthermore, we detected the uptake of exosomes by human umbilical vein endothelial cells (HUVECs) by immunofluorescence staining. In addition, we determined the effects of exosomes on cell viability, migration and tube formation in HUVECs by Cell Counting Kit‐8, migration and tube formation assays, respectively. We examined the expression of key proteins related to exosome‐induced angiogenesis by BMSCs cultured under hypoxic conditions by western blot. Exosomes released by BMSCs cultured under hypoxic conditions enhanced cell proliferation, migration and angiogenesis of HUVECs. Hypoxia induced the expression of high mobility group box 1 protein (HMGB1) in BMSC‐derived exosomes, and silencing of HMGB1 abolished the angiogenic effect in HUVECs. Furthermore, exosomal HMGB1 activated the JNK signaling pathway and induced hypoxia‐inducible factor‐1α/vascular endothelial growth factor expression, consequently enhancing angiogenesis in HUVECs. Our data reveal that exosomal HMGB1 promotes angiogenesis via JNK/hypoxia‐inducible factor‐1α signaling. Therefore, BMSC exosomes derived under hypoxia may have potential for development of novel treatment strategies for angiogenesis‐related diseases.

Characterizing Coagulation FVII from iPSC-Hepatocytes-like Cells: Setting the Basis for Cell Therapy Development

Introduction To date studies on factor (F)VII and other hepatic vitamin K-dependent coagulation factors have relied on cell lines overexpressing these human genes. Even though these models have provided insight into the biology of these factors, they do not fully illustrate the in vivo situation. Thus, a relevant physiological model that mimics the in vivo processing of FVII in liver cells with potential for therapeutic use is needed. Methods Human induced pluripotent stem cells (hiPSCs) were differentiated into hepatocyte-like cells (iHLCs) using a non-transcription factor based, small molecule approach. Cells were grown in medium with vitamin K to ensure a correct gamma-carboxylation. Cellular FVII mRNA and protein were determined by RT-qPCR and proteomic and Western blot (WB), respectively. Secreted FVII antigen was measured by ELISA and WB and FVII activity was assessed by chromogenic assay and thrombin generation assay (TGA). Post-translational modifications of FVII protein (glycosylation) were studied using digestion with N-glycosylase F (PNGase F) and neuraminidase. Confocal immunofluorescence microscopy was used to assess the cellular expression of FVII and other vitamin K- dependent coagulation factors and inhibitors. Human primary hepatocytes or human plasma pool were used as a control in the assays. Results The resulting iHLCs expressed FVII mRNA in comparable levels to primary hepatocytes and cellular FVII peptides were identified by mass spectrometry studies. iHLCs secreted FVII at levels of around 70% compared to primary hepatocytes with detectable activity around 35% of the FVII activity level from primary hepatocytes. The TGA showed that cell medium from iHLCs when mixed with FVII deficient plasma was able to induce thrombin generation faster than the FVII depleted plasma alone (lagtime 3.2 vs 27.6 s, respectively). PNGase-F treatment showed that FVII secreted by iHLCs was N-glycosylated. Intracellular FVII was detected by WB as a band of approximately 63 kDa, slightly larger than FVII from plasma pool but similar to FVII from primary hepatocytes. Moreover, additional coagulation factors and inhibitors such as FII, FX, protein C and antithrombin were detected both at the mRNA and protein levels in the cells. Conclusions Stem cell-derived iHLCs produce and secrete FVII at physiologically relevant levels. The resulting FVII showed similar post-translational modifications to plasma FVII although some differences in proteolysis could be inferred. This iHLCs-derived FVII is able to initiate the extrinsic coagulation pathway. Our data support that these iHLCs can serve as a highly relevant model to study FVII and other vitamin K-dependent coagulation factors in vitro and constitute an important step towards the development of novel cell-based therapies for both FVII and other vitamin K-dependent coagulation factor deficiencies. Disclosures No relevant conflicts of interest to declare.

Unconventional T Cells in the Pancreatic Tumor Microenvironment: Thinking Outside the Box

Unlike conventional MHC-reactive T cells, unconventional T cells have emerged as an abundant component of the human immune system because of their role in tumor immunology. In this issue of Cancer Discovery, Hundeyin and colleagues have identified a population of unconventional T cells in pancreatic tumors that can reprogram the immune evasive components of the tumor to promote immunogenicity and thus are critical for the development of novel cell-based therapy in pancreatic cancer.See related article by Hundeyin et al., p. 1288.

Comparative differentiation analysis of distinct oral tissue-derived cells in response to osteogenic stimulation.

Mixed cell populations from oral tissues may be superior to pure stem cells for regenerative approaches. Therefore, the aim of the present study was to explore the osteogenic potential of mixed cells derived from oral connective tissues compared to alveolar osteoblasts. Primary cells were isolated from the alveolar bone, periodontal ligament and gingiva. Following characterization by colony formation, growth capacity and flow cytometry, all cells were subjected to osteogenic differentiation induction and screened for a large panel of osteogenic markers using western blots, qPCR arrays, and matrix mineralization and alkaline phosphatase quantification. Non-induced mixed cells from gingiva showed higher colony formation efficiency but decreased proliferation compared to non-induced periodontal mixed cells, while both entities revealed similar surface markers tested in this setup. Following osteogenic induction, all cell populations individually expressed receptors with distinctively activated downstream effectors. Gene expression of induced periodontal mixed cells was similar to alveolar osteoblasts, but was differently modulated in gingival mixed cells. The latter failed to achieve osteogenic differentiation in terms of matrix mineralization and alkaline phosphatase activity, which was well observed in periodontal mixed cells and osteoblasts. Mixed cells from periodontal ligament but not from gingiva feature an inherent osteogenic capacity in vitro. From these results, it can be concluded that periodontal cells do not require further stem cell enrichment in order to qualify for bone regeneration. Our data contribute to the development of novel cell-based therapies using mixed cells from the periodontal ligament in regenerative periodontics.

Mechanisms of Cortical Differentiation.

During fetal and postnatal development, the human brain generates 160 billion neuronal and glial cells, each with precise cellular phenotypes. To effectively manage such a complicated task, intrinsic (e.g., transcription factors) and extrinsic (environmental signals) cues cooperate to regulate the decision by neural progenitors to continue to proliferate or to differentiate. Loss- and gain-of-function studies in the mouse brain have been instrumental in identifying these cues, leading to a fairly well-developed and well-integrated model of neocortical development. This research has revealed that the neurons, astrocytes, and oligodendrocytes that populate the mature neocortex are generated sequentially from neural progenitor pools in both the dorsal (pallial) and ventral (subpallial) telencephalon. Understanding how cellular diversity is established during neocortical development is critical, as appropriate numbers of inhibitory and excitatory neurons, oligodendrocytes, and astrocytes are required for normal neural function. Indeed, an imbalance in excitatory vs inhibitory neurotransmission or alterations in glial cell number are hallmark features of neuropsychological and intellectual disorders such as schizophrenia, bipolar disorder, and autism. Moreover, these fundamental studies are beginning to pave the way for the rational design of neural cell reprogramming strategies, which are of value for the assessment of disease etiology, and for the possible development of novel cell-based therapies. We review herein our current understanding of the intrinsic cues and environmental signals that govern cell fate specification and differentiation decisions during development of neuronal and glial lineages in the murine neocortex.

Stem cell-based therapies for tumors in the brain: are we there yet?

Advances in understanding adult stem cell biology have facilitated the development of novel cell-based therapies for cancer. Recent developments in conventional therapies (eg, tumor resection techniques, chemotherapy strategies, and radiation therapy) for treating both metastatic and primary tumors in the brain, particularly glioblastoma have not resulted in a marked increase in patient survival. Preclinical studies have shown that multiple stem cell types exhibit inherent tropism and migrate to the sites of malignancy. Recent studies have validated the feasibility potential of using engineered stem cells as therapeutic agents to target and eliminate malignant tumor cells in the brain. This review will discuss the recent progress in the therapeutic potential of stem cells for tumors in the brain and also provide perspectives for future preclinical studies and clinical translation.

Mesenchymal Stem Cell Based Therapies for Disc Repair/Regeneration

Low back pain (LBP) is one of the most common musculoskeletal disorders, with an estimated 84% of the population experiencing LBP at some point in their lifetime. The prevalence of LBP increases with age, suggesting incidences of LBP are likely to increase in the future due to a global aging population, changes in lifestyle and occupational stresses. Although the causes of LBP are multifactorial, increasing evidence implicates intervertebral disc (IVD) degeneration as a major contributor, with loss of IVD integrity leading to destabilization of the spinal motion segment, resulting in pain and disability. The IVD is a complex structure that allows movement between adjacent vertebrae and sustains the load applied through the spine. It consists of an outer annulus fibrosus (AF), a ligamentous lamellar structure composed predominantly of type I collagen fibers, and a central gelatinous nucleus pulposus (NP) composed predominantly of the proteoglycan aggrecan, interspersed with type II collagen fibers. In degeneration there is an alteration in NP cell biology leading to diminished cell numbers and altered cell function (largely increased catabolism) resulting in an imbalance between matrix synthesis and degradation, particularly within the NP. Current medical treatments for IVD degeneration rely on conservative therapies (e.g., pain relief, exercise therapy) and, when these fail, surgery. Surgical treatments such as spinal fusion and disc replacement have shown satisfactory results in alleviating pain, but are not devoid of complications and long-term clinical outcomes still remain poor. Thus, there is an urgent need for alternative therapies focused on correcting the underlying pathogenesis and aberrant cell biology of IVD degeneration. As such many researchers, including ourselves, are focusing on the development of novel cell-based therapies. However, in order for these to be successful an appropriate cell source for implantation, together with a suitable growth factor to direct cell differentiation and formation of a functional matrix formation must be identified. Additionally, extensive in vitro studies are needed to establish and support further pre-clinical and potential commercial development. Having characterized the phenotype of human NP cells (a prerequisite for tissue engineering/regenerative strategies to ensure correct differentiation of cells to the target native cell) we have applied this knowledge to demonstrate that both bone marrow MSCs (BM-MSCs) and adipose-derived MSCs (AD-MSCs) are capable of differentiation toward an NP-like phenotype. Specifically, we have demonstrated that stimulation of both BM-MSCs and AD-MSCs with GDF6 (compared with other members of the TGF-β superfamily) results in improved differentiation to an NP-like phenotype and, importantly, synthesis and deposition of an extracellular matrix, rich in proteoglycan and having micromechanical properties akin to the native healthy NP. Significantly, these studies have highlighted that AD-MSCs (rather than BM-MSCs) are the more appropriate cell source for NP regeneration and that GDF6 (alias CDMP-2 or BMP-13) is the most suitable growth factor for directing differentiation. Additionally we have shown that factors in the IVD niche, namely, hypoxia and load can modulate AD-MSC differentiation and that when these factors are combined they act synergistically to promote matrix formation and increase proteoglycan synthesis. For regeneration strategies, MSCs will be implanted into the degenerate IVD niche which is a milieu of catabolic and pro-inflammatory cytokines, particularly IL-1, and thus their response such pro-inflammatory factors needs to be ascertained to ensure that catabolic events are not exacerbated. Interestingly, when AD-MSCs that have been differentiated to NP-like cells (i.e., aNPCs) are exposed to IL-1, there is no significant fold changes in gene expression for the matrix molecules (ACAN, COL2A1), matrix degrading enzymes (MMP3, MMP13), or proteoglycan synthesis when compared with untreated aNPCs. This suggests that these cells may be able to withstand the effects of the catabolic milieu in the degenerate IVD niche. Furthermore, our studies have shown that GDF6 has anabolic effects on degenerate human NP cells, stimulating adoption of a more normal NP phenotype and increasing appropriate matrix synthesis. This suggests that delivery of GDF6 as part of an MSC-based regenerative therapy may be beneficial both in directing appropriate, lineage-specific MSC differentiation, but also in restoring a healthier, more anabolic phenotype in native NP cells, thereby having a dual regenerative effect. Importantly, these in vitro studies demonstrating that GDF6 promotes AD-MSC differentiation to NP cells and synthesis of an NP-like matrix, as well as potential effects of GDF6 on resident NP cells, suggests that our proposed combined biologic and cellular therapy can provide a significant step-change from existing interventions, potentially bridging the gap between symptomatic care and aggressive surgical interventions.

Intervertebral disc degeneration therapies in human

Low back pain (LBP) is one of the most common musculoskeletal disorders, with an estimated 84% of the population experiencing LBP at some point in their lifetime. As with most musculoskeletal disorders, the prevalence of LBP increases with age, suggesting incidences of LBP are likely to increase in the future due to a global aging population, changes in lifestyle and occupational stresses. Although the causes of LBP are multifactorial, increasing evidence implicates intervertebral disc (IVD) degeneration as a major contributor, with loss of IVD integrity leading to the destabilization of the spinal motion segment, resulting in pain and disability. The IVD is a complex structure that allows movement between adjacent vertebrae and sustains the load applied through the spine. It consists of the peripheral annulus fibrosus (AF), a ligamentous lamellar structure composed predominantly of type I collagen fibres, and the central nucleus pulposus (NP), a highly hydrated structure, composed of the proteoglycan aggrecan, interspersed with type II collagen fibres. Only 1% of the IVD volume is occupied by its constituent cells, but they assume a key role, as they maintain IVD homeostasis. In degeneration there is an alteration in NP cell biology leading to diminished cell numbers and altered cell function resulting in an imbalance between matrix synthesis and degradation, particularly within the NP. Current medical treatments for IVD degeneration rely on conservative therapies (e.g. pain relief, exercise therapy) and, when these fail, surgery. Surgical treatments such as spinal fusion and disc replacement have shown satisfactory results in alleviating pain, but are not devoid of complications and long-term clinical outcomes still remain poor. Thus, there is an urgent need for alternative therapies focussed on correcting the underlying pathogenesis and aberrant cell biology of IVD degeneration. As such many researchers, including ourselves, are focussing on the development of novel cell-based therapies. However, in order for these to be successful an appropriate cell source for implantation and tissue regeneration must be identified. In this presentation we discussed the pathophysiology of IVD degeneration, efforts to elucidate the phenotype of human IVD cells and how this has allowed development of mesenchymal stem cell (MSC)-based therapies for IVD regeneration. In particular it focused on our efforts to identify the optimal MSC source and growth factor to direct differentiation and enhance tissue formation, as well as the influence microenvironment has on regeneration strategies.

Combining perfluorocarbon and superparamagnetic iron‐oxide cell labeling for improved and expanded applications of cellular MRI

The ability to detect the migration of cells in living organisms is fundamental in understanding biological processes and important for the development of novel cell-based therapies to treat disease. MRI can be used to detect the migration of cells labeled with superparamagnetic iron-oxide (SPIO) or perfluorocarbon (PFC) agents. In this study, we explored combining these two cell-labeling approaches to overcome current limitations and enable new applications for cellular MRI. We characterized (19)F-NMR relaxation properties of PFC-labeled cells in the presence of SPIO and imaged cells both ex vivo and in vivo in a rodent inflammation model to demonstrate selective visualization of cell populations. We show that with UTE3D, RARE, and FLASH (19) F images one can uniquely identify PFC-labeled cells, colocalized PFC- and SPIO-labeled cells, and PFC/SPIO-colabeled cells. This new methodology has the ability to improve and expand applications of MRI cell tracking. Combining PFC and SPIO strategies can potentially provide a method to quench PFC signal transferred from dead cells to macrophages, thereby eliminating false positives. In addition, combining these techniques could also be used to track two cell types simultaneously and probe cell-cell proximity in vivo with MRI.

Effect of cell culture using chitosan membranes on stemness marker genes in mesenchymal stem cells

Mesenchymal stem cell (MSC) therapy is a promising treatment for diseases of the nervous system. However, MSCs often lose their stemness and homing abilities when cultured in conventional two‑dimensional (2D) systems. Consequently, it is important to explore novel culture methods for MSC-based therapies in clinical practice. To investigate the effect of a cell culture using chitosan membranes on MSCs, the morphology of MSCs cultured using chitosan membranes was observed and the expression of stemness marker genes was analyzed. We demonstrated that MSCs cultured using chitosan membranes form spheroids. Additionally, the expression of stemness marker genes, including Oct4, Sox2 and Nanog, increased significantly when MSCs were cultured using chitosan membranes compared with 2D culture systems. Finally, MSCs cultured using chitosan membranes were found to have an increased potential to differentiate into nerve cells and chrondrocytes. In conclusion, we demonstrated that MSCs cultured on chitosan membranes maintain their stemness and homing abilities. This finding may be further investigated for the development of novel cell-based therapies for diseases involving neuron-like cells and chondrogenesis.

Editorial introductions

Editorial introductions

Identification of rare progenitor cells from human periosteal tissue using droplet microfluidics

The isolation and characterisation of single cells from a heterogeneous population are important processes in cell biology, immunology, stem cell research, and cancer research. In the development of novel cell-based therapies, there is a considerable need to target specific cell types to allow for further analysis and amplification ex vivo. We introduce, herein, the use of droplet-based microfluidics as a platform technology for the identification and quantification of distinct cell phenotypes. Using molecular labelling of specific cell populations by antibodies and fluorescent dyes, detection of single cells encapsulated within picolitre-sized aqueous droplets can be performed using high-sensitivity confocal fluorescence detection. Specifically, rare progenitor cells were immunodetected within a heterogeneous population of cells isolated from human periosteal tissue. Using this model human cell population, the accuracy and reproducibility of the droplet system were tested and the results were verified using conventional flow cytometry. It was found that the quantitation of phenotypic subpopulations measured using both techniques is directly comparable. Accordingly, this study demonstrates the biological capacity of droplet-based microfluidics for cellular analysis and provides a necessary first step towards the development of a novel cell sorting technology.

Potentials of ES Cell Therapy in Neurodegenerative Diseases

Neurodegenerative diseases comprise a heterogeneous spectrum of neural disorders that cause severe and progressive cognitive and motor deficits. A histological hallmark of these disorders is the occurrence of disease-specific cell death in specific regional subpopulations of neurons, such as the loss of dopaminergic neurons in the substantia nigra in Parkinson's disease. Neurodegenerative disease can also possibly occur from the loss or dysfunction of selected glial cell subsets, such as the dysfunction of supportive glial cells around somatic motor neurons in amyotrophic lateral sclerosis. The central nervous system (CNS), unlike many other tissues, has a very limited capacity for self-repair. Mature nerve cells lack the ability to regenerate, although endogenous neural stem cells exist in the adult brain that do have very limited ability to generate new functional neurons in response to injury. Rapid advances in stem cell biology have opened an alternative, fascinating perspective of neurogenesis by activation of endogenous neural stem cells and/or transplantation of in vitro-expanded stem cells and/or their neuronal- or glial-differentiated progeny. Embryonic stem (ES) cells, because of their ability to provide seemingly unlimited supply of specific cell types, their amenability to genetic engineering manipulations, and their broad developmental potential, are expected to become a cell source and biological delivery system for use in a variety of neurodegenerative diseases, and are likely to play a role in the development of novel cell-based therapies for these indications. However, before the full potential of ES cells can be realized for regenerative medicine, we need to understand mechanisms regulating their proliferation, differentiation into therapeutically relevant cells, and most importantly in the case of neuronal and glial lineages, to characterize their functional properties. In the present review we will be focusing on the factors and methodologies responsible for differentiation of ES cell into different neural precursors and neural cell lineages with particular emphasis on the potential research and clinical applications of ES cells in the field of neurodegenerative disease.

R410 – Immunosuppressive Properties of Tonsil-Derived MSCs

Problem We have previously shown that stroma of human palatine tonsils contains mesenchymal stem cells (MSCs) that can be isolated and expanded in culture. These tonsil-derived MSCs (T-MSCs) show multipotent differentiation properties, i.e. can differentiate along multiple mesenchymal lineages, including osteoblasts, chondrocytes, adipocytes, and myocytes. Recent findings also show that MSCs display immunoregulatory properties. Although the exact immunosuppressive mechanisms are unknown, the capacity of MSCs to suppress T-cell proliferation stimulated by allogeneic lymphocytes, dendritic cells, and phytohemaglutinin (PHA) is well documented. This study explores immunosuppressive characteristics of T-MSCs and compares them with characteristics of bone marrow-derived MSCs (BM-MSCs), a well-characterized cell population. Methods The mixed lymphocyte reaction (MLR) using human peripheral blood mononuclear cells (PBMC) from healthy donors and xenogeneic murine splenocytes was used to test the immunosuppressive properties of T-MSCs and BMMSCs. Indoleamine 2,3-dioxygenase (IDO) enzyme activity was measured spectrophotometrically based on tryptophan-to-kynurenine conversion in the supernatant. Interferon (IFN)-g in culture supernatants was quantified using a commercially available ELISA kit. Results Addition of BM-MSCs and T-MSCs both inhibited the PHA-induced proliferative response of PBMC and xenogeneic splenocytes. The difference in immunosuppressive activity correlates with the level of cell surface interferon (IFN)-g receptor as well as the differential ability of IFN-g to stimulate of IDO activity by T-MSCs compared to BM-MSCs. Conclusion T-MSCs share similar immunosuppressive characteristics as BM-MSCs in MLR. The immunosuppressive activity is significant and dose-dependent, although at a lower level than that of BM-MSCs. Significance Owing to their ease of isolation, rapid proliferation in the culture and self-renewal capacity, MSCs to date are considered an attractive candidate cell type for the development of novel cell-based therapies. They could be relevant in a wide range of clinical applications, including tissue and organ repair, drug or gene delivery to diseased tissues, improvement of allogenic hematopoietic cell transplantation, and the management of graft-versus-host disease. Support Supported by NIAMS Intramural Research Program (NIH ZO1 AR 41131). Sasa Janjanin is a recipient of the Fulbright Scholarship of the U.S. Department of State.

Molecular analysis of early host cell infection by Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas heart disease, infects heart and other cells leading to cardiac arrest frequently followed by death. The disease affects millions of individuals in the Americas and is posing health problems because of blood transmission in the US due to large Latin American immigration. Since the current drugs present serious side effects and do not cure the chronic infection, it is critically important to understand the early process of cellular infection at the molecular and structural levels to design novel inhibitors to block T. cruzi infection. In this review, the authors critically analyze the molecular and cellular basis of early T. cruzi infection and discuss the future directions in this area. The candidate T. cruzi invasive genes and host genes involved in the process of early infection are just beginning to be understood. The trypanosome invasive proteins are excellent targets for intervention. The progress made in the cell biology of T. cruzi infection will also facilitate the development of novel cell-based therapies to ameliorate the disease.

Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells

The development of novel cell-based therapies requires understanding of distinct human hematopoietic stem and progenitor cell populations. We recently isolated reconstituting hematopoietic stem cells (HSCs) by lineage depletion and purification based on high aldehyde dehydrogenase activity (ALDH(hi)Lin- cells). Here, we further dissected the ALDH(hi)-Lin- population by selection for CD133, a surface molecule expressed on progenitors from hematopoietic, endothelial, and neural lineages. ALDH(hi)CD133+Lin- cells were primarily CD34+, but also included CD34-CD38-CD133+ cells, a phenotype previously associated with repopulating function. Both ALDH(hi)CD133-Lin- and ALDH(hi)CD133+Lin- cells demonstrated distinct clonogenic progenitor function in vitro, whereas only the ALDH(hi)CD133+Lin- population seeded the murine bone marrow 48 hours after transplantation. Significant human cell repopulation was observed only in NOD/SCID and NOD/SCID beta2M-null mice that received transplants of ALDH(hi)CD133+Lin- cells. Limiting dilution analysis demonstrated a 10-fold increase in the frequency of NOD/SCID repopulating cells compared with CD133+Lin- cells, suggesting that high ALDH activity further purified cells with repopulating function. Transplanted ALDH(hi)CD133+Lin- cells also maintained primitive hematopoietic phenotypes (CD34+CD38-) and demonstrated enhanced repopulating function in recipients of serial, secondary transplants. Cell selection based on ALDH activity and CD133 expression provides a novel purification of HSCs with long-term repopulating function and may be considered an alternative to CD34 cell selection for stem cell therapies.

Prospects for the Xenopus Embryo Model in Therapeutics Technologies

Access to suitable animal models is essential in the field of therapeutics technologies. Recently, lower vertebrates have emerged as attractive low-cost animal models which offer new exciting applications in functional genomics and therapeutics technologies. Amphibian embryos of the genus Xenopus have long served as important models for the study of vertebrate development. Xenopus is evolutionary significantly less distant to humans than fish models, which suggests that experimental findings gained with Xenopus will more accurately predict human biology. Numerous experimental advantages, including external development, large size, identifiable blastomeres, and their ability to withstand extensive surgical intervention and culture in vitro, have favored the use of the Xenopus model in the past. More recently, the introduction of a simple efficient method to disrupt gene functions and the rapid development of genomic resources has further increased the attractiveness of this low-cost, high-throughput model for the analysis of vertebrate gene functions. Using the Xenopus embryo as the primary animal model, our research in the field of therapeutics technologies has focused on the identification and validation of novel drug targets by employing genomic and transcriptomic information in the analysis of the molecular and cellular processes underlying kidney organogenesis and vascular development. Furthermore, our research on signaling pathways controlling cellular differentiation of embryonic tissues provides important insights that may ultimately lead to the development of novel cell-based therapies in regenerative medicine. Finally, we are exploring the possibility of employing the Xenopus embryos in chemical library screens to identify novel chemical modulators of organogenesis.