The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration

Planarian flatworms are an exception among bilaterians in that they possess a large pool of adult stem cells that enables them to promptly regenerate any part of their body, including the brain. Although known for two centuries for their remarkable regenerative capabilities, planarians have only recently emerged as an attractive model for studying regeneration and stem cell biology. This revival is due in part to the availability of a sequenced genome and the development of new technologies, such as RNA interference and next-generation sequencing, which facilitate studies of planarian regeneration at the molecular level. Here, we highlight why planarians are an exciting tool in the study of regeneration and its underlying stem cell biology in vivo, and discuss the potential promises and current limitations of this model organism for stem cell research and regenerative medicine.

Self-Renewing Human Bone Marrow Mesenspheres Promote Hematopoietic Stem Cell Expansion.

Strategies for expanding hematopoietic stem cells (HSCs) include coculture with cells that recapitulate their natural microenvironment, such as bone marrow stromal stem/progenitor cells (BMSCs). Plastic-adherent BMSCs may be insufficient to preserve primitive HSCs. Here, we describe a method of isolating and culturing human BMSCs as nonadherent mesenchymal spheres. Human mesenspheres were derived from CD45- CD31- CD71- CD146+ CD105+ nestin+ cells but could also be simply grown from fetal and adult BM CD45--enriched cells. Human mesenspheres robustly differentiated into mesenchymal lineages. In culture conditions where they displayed a relatively undifferentiated phenotype, with decreased adherence to plastic and increased self-renewal, they promoted enhanced expansion of cord blood CD34+ cells through secreted soluble factors. Expanded HSCs were serially transplantable in immunodeficient mice and significantly increased long-term human hematopoietic engraftment. These results pave the way for culture techniques that preserve the self-renewal of human BMSCs and their ability to support functional HSCs.

Peripheral Nerve Repair: Multimodal Comparison of the Long-Term Regenerative Potential of Adipose Tissue-Derived Cells in a Biodegradable Conduit.

Tissue engineering is a popular topic in peripheral nerve repair. Combining a nerve conduit with supporting adipose-derived cells could offer an opportunity to prevent time-consuming Schwann cell culture or the use of an autograft with its donor site morbidity and eventually improve clinical outcome. The aim of this study was to provide a broad overview over promising transplantable cells under equal experimental conditions over a long-term period. A 10-mm gap in the sciatic nerve of female Sprague-Dawley rats (7 groups of 7 animals, 8 weeks old) was bridged through a biodegradable fibrin conduit filled with rat adipose-derived stem cells (rASCs), differentiated rASCs (drASCs), human (h)ASCs from the superficial and deep abdominal layer, human stromal vascular fraction (SVF), or rat Schwann cells, respectively. As a control, we resutured a nerve segment as an autograft. Long-term evaluation was carried out after 12 weeks comprising walking track, morphometric, and MRI analyses. The sciatic functional index was calculated. Cross sections of the nerve, proximal, distal, and in between the two sutures, were analyzed for re-/myelination and axon count. Gastrocnemius muscle weights were compared. MRI proved biodegradation of the conduit. Differentiated rat ASCs performed significantly better than undifferentiated rASCs with less muscle atrophy and superior functional results. Superficial hASCs supported regeneration better than deep hASCs, in line with published in vitro data. The best regeneration potential was achieved by the drASC group when compared with other adipose tissue-derived cells. Considering the ease of procedure from harvesting to transplanting, we conclude that comparison of promising cells for nerve regeneration revealed that particularly differentiated ASCs could be a clinically translatable route toward new methods to enhance peripheral nerve repair.

Reticular dysgenesis-associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress.

Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.

An Advocacy for the Use of 3D Stem Cell Culture Systems for the Development of Regenerative Medicine: An Emphasis on Photoreceptor Generation

The availability of stem cells is of great promise to study early developmental stages and to generate adequate cells for cell transfer therapies. Although many researchers using stem cells were successful in dissecting intrinsic and extrinsic mechanisms and in generating specific cell phenotypes, few of the stem cells or the differentiated cells show the capacity to repair a tissue. Advances in cell and stem cell cultivation during the last years made tremendous progress in the generation of bona fide differentiated cells able to integrate into a tissue after transplantation, opening new perspectives for developmental biology studies and for regenerative medicine. In this review, we focus on the main works attempting to create in vitro conditions mimicking the natural environment of CNS structures such as the neural tube and its development in different brain region areas including the optic cup. The use of protocols growing cells in 3D organoids is a key strategy to produce cells resembling endogenous ones. An emphasis on the generation of retina tissue and photoreceptor cells is provided to highlight the promising developments in this field. Other examples are presented and discussed, such as the formation of cortical tissue, the epithelial gut or the kidney organoids. The generation of differentiated tissues and well-defined cell phenotypes from embryonic stem (ES) cells or induced pluripotent cells (iPSCs) opens several new strategies in the field of biology and regenerative medicine. A 3D organ/tissue development in vitro derived from human cells brings a unique tool to study human cell biology and pathophysiology of an organ or a specific cell population. The perspective of tissue repair is discussed as well as the necessity of cell banking to accelerate the progress of this promising field.

Enhancement of human adipose-derived stem cell expansion and stability for clinical use

Co-culture techniques associating both dermal fibroblasts and epidermal keratinocytes have shown to have better clinical outcome than keratinocyte culture alone for the treatment of severe burns. Since fat grafting has been shown to improve scar remodelling, new techniques such as cell-therapy-assisted surgical reconstruction with isolated and expanded autologous adipose-derived stem cells (ASCs) would be of benefit to increase graft acceptation. Therefore, integrating ASCs into standardized procedures for cultured skin grafting could be of benefit for the patient if cell quality and quantity could be maintained. The purpose of this study was to evaluate ASC processing from adult tissue with simple isolation (without enzymatic steps), expansion (low density of 325-3,000 cells/cm2) and storage conditions to assure methods to enhance the cellular resistance when transferred back to the patient. Co-culture with cell-banked skin progenitor cells (FE002-SK2) showed an increase of 40-50% ASCs yield at high passages alongside with a better preservation of morphology, proper adipogenic and osteogenic differentiation and efficient biocompatibility with 3D collagen scaffolds. ASCs can be considered as a valuable additional cell source to be delivered in biological bandages to the patient in a need of tissue reconstruction such as burn patients.

IMP2 provides an alternative to LIN28B for LET-7 target gene protection and glioma stem cell maintenance

Glioblastoma multiforme (GBM) is the most frequent and lethal primary brain tumor in adults. Accumulating evidence suggests that tumors comprise a hierarchical organization that is, at least partially, not genetically driven. Cells that reside at the apex of this hierarchy are commonly referred to as cancer stem cells (CSCs) and are believed to largely contribute to recurrence and therapeutic failure. Although the complexity of epigenetic regulation of the genome precludes prediction as to which epigenetic changes dominate CSC specification in different cancer types, the ability of microRNAs (miRNAs) to fine-tune expression of entire gene networks places them among prime candidates for establishing CSC properties. In this study we characterized the miRNA expression profile of primary GBM grown either under conditions that enrich for GSCs or their differentiated non-tumorigenic progeny (DGCs). Although, we identified a subset of miRNAs that was strongly differentially expressed between GSCs and DGCs, we observed that in GSCs both let-7 and, paradoxically, their target genes are highly expressed, suggesting protection against let-7 action. Using PAR-CLIP we show that insulin-like growth factor-2 mRNA-binding protein 2 (IMP2) provides a mechanism for let-7 target gene protection that represents an alternative to LIN28A/B, which abrogates let-7 biogenesis in normal embryonic and certain malignant stem cells. By direct binding to miRNA recognition elements, IMP2 protects its targets from let-7 mediated decay. Importantly, depletion of IMP2 in GSCs strongly impairs their self- renewal properties and tumorigenicity in vivo, a phenotype that can be rescued by expression of LIN28B, suggesting that IMP2 mainly contributes to GSC maintenance by protecting let-7 target genes from silencing. Using mouse models, we show that depletion of IMP2 in neural stem cells (NSCs) induces let-7 target gene down-regulation, impairs their clonogenic capacity, and affects differentiation. Taken together, our observations describe a novel regulatory function of IMP2 in the let-7 axis whereby it supports GSC and NSC specification. Résumé (Français) Le glioblastome (GBM) est la tumeur primaire maligne du cerveau la plus fréquente. De nombreuses études ont démontré l'existence d'une organisation hiérarchique des cellules cancéreuses liée à des mécanismes épigénétiques. Les cellules qui se trouvent au sommet de cette hiérarchie sont appelées cellules souches cancéreuses (CSC), et contribuent à l'échec thérapeutique. Bien que la complexité des régulateurs épigénétiques permette difficilement de prédire quel mécanisme contribue le plus aux propriétés des CSC, la capacité des microRNAs (miRNAs) de réguler des réseaux entiers de gènes, les placent comme des candidats de premiers choix. Ici, nous avons caractérisé le profil d'expression des miRNAs dans des tumeurs primaires de GBM cultivées dans des conditions qui enrichissent soit pour les CSC, soit pour leur contrepartie de cellules cancéreuses différences (CCD). De manière surprenante et paradoxale la famille de miRNA let-7 et leurs gènes cibles étaient hautement exprimés dans les CSC, suggérant un mécanisme de protection contre l'action des let-7. Avec l'aide de la technologie PAR-CLIP, nous démontrons que la protéine IMP2, protège les mRNAs de l'action des let-7 et représente une alternative à Lin28A/B, qui d'ordinaire réprime fortement la maturation des let-7 dans les cellules souches embryonnaires et divers cancers. En se liant à la région ciblée par les let-7, IMP2 protège ses transcrits de l'action de cette classe de microRNA qui est tumoro-supressive. La déplétion d'IMP2 dans des CSC de GBM réduit fortement leur clonogénicité in vitro et leur tumorigénicité in vivo. Ceci peut être reversé en introduisant Lin28B dans des CSC de GBM, suggérant qu'IMP2 exerce ses fonctions pro-tumorigéniques en modulant l'axe let-7. Avec l'aide de modèles murins, nous observons que la déplétion de IMP2 dans les cellules souches neurales (CSN) induit une baisse de leur clonogénicité et des cibles des miRNAs let-7, suggérant une conservation de ce mécanisme entre les CSC de GBM et les CSN. En résumé, nos observations définissent une nouvelle fonction de IMP2 dans l'axe let-7 par lequel il contribue au maintien des propriétés des CSC et des CSN.

Circulating progenitor cells during exercise, muscle electro-stimulation and intermittent hypobaric hypoxia in patients with traumatic brain injury. A pilot study

BACKGROUND: Circulating progenitor cells (CPC) treatments may have great potential for the recovery of neurons and brain function. OBJECTIVE: To increase and maintain CPC with a program of exercise, muscle electro-stimulation (ME) and/or intermittent-hypobaric-hypoxia (IHH), and also to study the possible improvement in physical or psychological functioning of participants with Traumatic Brain Injury (TBI). METHODS: Twenty-one participants. Four groups: exercise and ME group (EEG), cycling group (CyG), IHH and ME group (HEG) and control group (CG). Psychological and physical stress tests were carried out. CPC were measured in blood several times during the protocol. RESULTS: Psychological tests did not change. In the physical stress tests the VO2 uptake increased in the EEG and the CyG, and the maximal tolerated workload increased in the HEG. CPC levels increased in the last three weeks in EEG, but not in CyG, CG and HEG. CONCLUSIONS: CPC levels increased in the last three weeks of the EEG program, but not in the other groups and we did not detect performed psychological test changes in any group. The detected aerobic capacity or workload improvement must be beneficial for the patients who have suffered TBI, but exercise type and the mechanisms involved are not clear.

Autologous Stem Cell Transplantation In Multiple Myeloma

Background. Multiple myeloma (MM) is the second most common hematologic malignancy after lymphomas In Finland: the annual incidence of MM is approximately 200. For three decades the median survival remained at 3 to 4 years from diagnosis until high-dose melphalan treatment supported by autologous stem cell transplantation (ASCT) became the standard of care for newly diagnosed MM since the mid 1990’s and the median survival increased to 5 – 6 years. This study focuses on three important aspects of ASCT, namely 1) stem cell mobilization, 2) single vs. double ASCT as initial treatment, and 3) the role of minimal residual disease (MRD) for longterm outcome. Aim. The aim of this series of studies was to evaluate the outcomes of MM patients and the ASCT procedure at the Turku University Central Hospital, Finland. First, we tried to identify which factors predict unsuccessful mobilization of autologous stem cells. Second, we compared the use of short-acting granulocyte-colony stimulating factor (GCSF) with long-acting G-CSF as mobilization agents. Third, one and two successive ASCTs were compared in 100 patients with MM. Fourth, for patients in complete response (CR) after stem cell transplantation (SCT), patient-specific probes for quantitative allele-specific oligonucleotide polymerase-chain reaction (qASO-PCR) measurements were designed to evaluate MRD and its importance for long-term outcome. Results. The quantity of previous chemotherapy and previous interferon use were significant pre-mobilization factors that predicted mobilization failure, together with some factors related to mobilization therapy itself, such as duration and degree of cytopenias and occurrence of sepsis. Short-acting and long-acting G-CSF combined with chemotherapy were comparable as stem cells mobilizers. The progression free (PFS) and overall survival (OS) tended to be longer after double ASCT than after single ASCT with a median follow-up time of 4 years, but this difference disappeared as the follow-up time increased. qASO-PCR was a good and sensitive divider of the CR patients into two prognostic groups: MRD low/negative (≤ 0.01%) and MRD high (>0.01%) groups with a significant difference in PFS and suggestively also in OS. Conclusions. When the factors prediciting a poor outcome of stem cell mobilization prevail, it is possible to identify those patients who need specific efforts to maximize the mobilization efficacy. Long-acting pegfilgrastim is a practical and effective alternative to short-acting filgrastim for mobilization therapy. There is no need to perform double ASCT on all eligible patients. MRD assessment with qASO-PCR is a sensitive method for evaluation of the depth of the CR response and can be used to predict long-term outcome after ACST.

Isolation and characterization of equine peripheral blood-derived multipotent mesenchymal stromal cells

The objective of the study was to isolate, cultivate and characterize equine peripheral blood-derived multipotent mesenchymal stromal cells (PbMSCs). Peripheral blood was collected, followed by the isolation of mononuclear cells using density gradient reagents, and the cultivation of adherent cells. Monoclonal mouse anti-horse CD13, mouse anti-horse CD44, and mouse anti-rat CD90 antibodies were used for the immunophenotypic characterization of the surface of the PbMSCs. These cells were also cultured in specific media for adipogenic and chondrogenic differentiation. There was no expression of the CD13 marker, but CD44 and CD90 were expressed in all of the passages tested. After 14 days of cell differentiation into adipocytes, lipid droplets were observed upon Oil Red O (ORO) staining. Twenty-one days after chondrogenic differentiation, the cells were stained with Alcian Blue. Although the technique for the isolation of these cells requires improvement, the present study demonstrates the partial characterization of PbMSCs, classifying them as a promising type of progenitor cells for use in equine cell therapy.

Protocol for collection and separation of bone marrow mononuclear cells in Chlorocebus aethiops

Abstract: Chlorocebus aethiops is a species of non-human primate frequently used in biomedical research. Some research involves this species as an experimental model for various diseases and possible treatment with stem cells. The bone marrow is one of the main sources of these cells and provides easy access. The aim of this study was to standardize the protocol of collection and separation of bone marrow in C. aethiops. Ten animals were submitted to puncture of bone marrow with access to the iliac crest and cell separation by density gradient. The bone marrow of C. aethiops had an average of 97% viability. From the results achieved, we can conclude that C. aethiops is an excellent model to obtain and isolate mononuclear cells from bone marrow, fostering several studies in the field of cell therapy.

New strategies in hematopoietic stem cell transplantation: G-CSF-mobilized unprocessed whole blood

Transplantation of mobilized peripheral blood stem cells (PBSC) for rescue of bone marrow function after high-dose chemo-/radiotherapy is widely used in hematologic malignancies and solid tumors. Mobilization of stem cells to the peripheral blood can be achieved by cytokine treatment of the patients. The main advantage of autologous PBSC transplantation over bone marrow transplantation is the faster recovery of neutrophil and platelet counts. The threshold number of PBSC required for adequate rescue of bone marrow is thought to be about 2 x 106 CD34+ cells/kg, if the stem cells are collected by leukapheresis and subsequently cryopreserved. We show that this critical number could be further reduced to as few as 0.2 x 106 cells/kg. In 30 patients with multiple myeloma and 25 patients with bad risk lymphoma 1 liter of granulocyte colony-stimulating factor (G-CSF)-mobilized unprocessed whole blood (stored at 4oC for 1-3 days) was used for transplantation. Compared to a historical control group, a significant reduction in the duration of neutropenia, thrombocytopenia and the length of hospital stay was documented. Furthermore, the effect of stem cell support was reflected by a lower need for platelet and red cell transfusions and a reduced antibiotic use. Considering the data as a whole, a cost saving of about 50% was achieved. To date, this easy to perform method of transplantation is only feasible following high-dose therapies that are completed within 72 h, since longer storage of unprocessed blood is accompanied by a substantial loss of progenitor cell function. Ongoing investigations include attempts to prolong storage times for whole blood

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells

The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (10(6) cells) obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5) were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs) were also labeled with tetramethylrhodamine-labeled alpha-bungarotoxin. The development of ectopic muscles was successful in 86% of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4), resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. The presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 ± 1.0 µm; N = 213), the diameter of ectopic fibers (59.1 µm; N = 213) did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.

The role of osteoblasts in regulating hematopoietic stem cell activity and tumor metastasis

Bone marrow stromal cells are critical regulators of hematopoiesis. Osteoblasts are part of the stromal cell support system in bone marrow and may be derived from a common precursor. Several studies suggested that osteoblasts regulate hematopoiesis, yet the entire mechanism is not understood. It is clear, however, that both hematopoietic precursors and osteoblasts interact for the production of osteoclasts and the activation of resorption. We observed that hematopoietic stem cells (HSCs) regulate osteoblastic secretion of various growth factors, and that osteoblasts express some soluble factors exclusively in the presence of HSCs. Osteoblasts and hematopoietic cells are closely associated with each other in the bone marrow, suggesting a reciprocal relationship between them to develop the HSC niche. One critical component regulating the niche is stromal-derived factor-1 (SDF-1) and its receptor CXCR4 which regulates stem cell homing and, as we have recently demonstrated, plays a crucial role in facilitating those tumors which metastasize to bone. Osteoblasts produce abundant amounts of SDF-1 and therefore osteoblasts play an important role in metastasis. These findings are discussed in the context of the role of osteoblasts in marrow function in health and disease.