Human Hemopoietic Cells Research Articles

Tetraspanin CD53 controls Tcell immunity through regulation of CD45RO stability, mobility, and function.

T cells depend on the phosphatase CD45 to initiate Tcell receptor signaling. Although the critical role of CD45 in Tcells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in Tcell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and Tcell activation. CD53-negative Tcells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing Tcells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for Tcell immunity.

A Novel Model of Human Hemopoiesis Based on Single Cell Functional and Transcriptional Analysis of Lympho-Myeloid Progenitors

Introduction: Human hemopoiesis produces 10 billion new, terminally mature blood cells daily; a production that is also rapidly responsive to external stimuli. Dysregulation of this complex process can lead to hemopoietic and immune deficiencies and blood cancers. In humans, the hemopoietic progenitor hierarchy producing lymphoid and myeloid lineages is unclear. Multiple progenitor populations give rise to lymphoid and myeloid cells but they remain incompletely characterized at the immunophenotypic, transcriptional and functional level. Here, we aim to understand the clonal functional output and transcriptional programs of primary human lympho-myeloid progenitor populations - the lymphoid-primed multipotent progenitor (LMPP) (Goardon et al., Cancer Cell, 2011), the multi-lymphoid progenitor (MLP) (Doulatov et al., Nature Immunology, 2010) and the granulocyte-macrophage progenitor (GMP).Methods: We devised a FACS strategy to prospectively purify eight human hemopoietic stem and progenitor cell populations. We functionally compared the LMPP, MLP and GMP in vitro by quantitative CFU assays, three different single cell liquid cultures or limit dilution analysis and in vivo by transplantation into a humanized ossicle mouse model (Reinisch et al., Nature Medicine, 2016). We performed population and single cell RNA sequencing and Fluidigm qRT-PCR to understand the relationship between the functional and transcriptional heterogeneity within human lympho-myeloid progenitor populations.Results: Our study comprehensively characterized the LMPP, MLP and GMP lympho-myeloid progenitor populations. Both LMPP and MLP are very rare within the mononuclear fraction (1 in 104 and 1 in 105 cells in CB and BM, respectively). We cultured 4357 single LMPP, GMP and MLP cells (isolated from 25 cord blood units and equivalent to ~1010 mononuclear cells) under three different culture conditions, one of which is described for the first time. We observed marked functional heterogeneity in the three lympho-myeloid progenitor populations. Focusing on the wells that gave single cell-initiated cultures, the majority of cells from LMPP, MLP and GMP gave uni-lineage output (60-90%). Bi-lineage output was less common (up to 35%), while multi-lineage output was the least frequent and only observed from LMPP and GMP cells (up to 15%). In vivo transplantation using the humanized ossicle mouse model increased the engraftment of lympho-myeloid progenitors 10-fold compared to previous reports. In vivo, the LMPP and GMP gave robust engraftment but the MLP substantially less engraftment. The LMPP gave rise to both myeloid and B cell engraftment, the GMP to myeloid engraftment and the MLP mainly to B cell output. Population RNA sequencing revealed distinct transcriptional signatures of these populations: MLP signature was enriched for lymphoid-affiliated genes, GMP for myeloid-affiliated genes and LMPP had a hybrid lympho-myeloid signature. Single cell RNA sequencing of 320 LMPP, MLP and GMP cells showed that the 3 progenitor populations form a transcriptional continuum. Linking the single cell gene expression to FACS indexing allowed exclusive purification of lymphoid- and myeloid-only progenitors from the current LMPP and GMP populations.Conclusions: These data change our understanding of human hematopoiesis and offer a radically new model of human lympho-myeloid progenitor specification. This model may have important implications for human immune deficiencies and hemopoietic malignancies.BS and DK contributed equally to this work. FH and AR contributed equally to this work. DisclosuresQuek:Celgene Corporation: Research Funding. Vyas:Jazz Pharmaceuticals: Speakers Bureau; Celgene Corporation: Speakers Bureau.

Human CD34+ Hematopoietic Stem Cell–Engrafted NSG Mice: Morphological and Immunophenotypic Features

Immunodeficient mice engrafted with human immune cells represent an innovative tool to improve translatability of animal models for the study of human diseases. Immunophenotyping in these mice focuses on engraftment rates and cellular differentiation in blood and secondary lymphoid organs, and is predominantly carried out by FACS (fluorescent activated cell sorting) analysis; information on the morphological aspects of engraftment and the prevalence of histologic lesions is limited. We histologically examined 3- to 6-month-old NSG mice, naïve or engrafted with CD34+ human hemopoietic stem cells (HSC), and employed a quantitative immunohistochemical approach to identify human and murine cell compartments, comparing the results with the FACS data. NSG mice mainly exhibited incidental findings in lungs, kidneys, testes, and adrenal glands. A 6-month-old NSG mouse had a mediastinal lymphoblastic lymphoma. The lymphoid organs of NSG mice lacked typical lymphoid tissue architecture but frequently exhibited small periarteriolar leukocyte clusters in the spleen. Mice engrafted with human HSC frequently showed nephropathy, ovarian atrophy, cataract, and abnormal retinal development, lesions considered secondary to irradiation. In addition, 20% exhibited multisystemic granulomatous inflammatory infiltrates, dominated by human macrophages and T cells, leading to the observed 7% mortality and morbidity. Immunophenotypic data revealed variable repopulation of lymphoid organs with hCD45+ human cells, which did not always parallel the engraftment levels measured via FACS. The study describes the most common pathological features in young NSG mice after human HSC engraftment. As some of these lesions contribute to morbidity, morphological assessment of the engraftment at tissue level might help improve immunophenotypic evaluations of this animal model.

By reducing global mRNA translation in several ways, 2-deoxyglucose lowers MCL-1 protein and sensitizes hemopoietic tumor cells to BH3 mimetic ABT737

Drugs targeting various pro-survival BCL-2 family members (‘‘BH3 mimetics’’) have efficacy in hemopoietic malignancies, but the non-targeted pro-survival family members can promote resistance. Pertinently, the sensitivity of some tumor cell lines to BH3 mimetic ABT737, which targets BCL-2, BCL-XL, and BCL-W but not MCL-1, is enhanced by 2-deoxyglucose (2DG). We found that 2DG augmented apoptosis induced by ABT737 in 3 of 8 human hemopoietic tumor cell lines, most strongly in pre-B acute lymphocytic leukemia cell line NALM-6, the focus of our mechanistic studies. Although 2DG can lower MCL-1 translation, how it does so is incompletely understood, in part because 2DG inhibits both glycolysis and protein glycosylation in the endoplasmic reticulum (ER). Its glycolysis inhibition lowered ATP and, through the AMPK/mTORC1 pathway, markedly reduced global protein synthesis, as did an ER integrated stress response. A dual reporter assay revealed that 2DG impeded not only cap-dependent translation but also elongation or cap-independent translation. MCL-1 protein fell markedly, whereas 12 other BCL-2 family members were unaffected. We ascribe the MCL-1 drop to the global fall in translation, exacerbated for mRNAs with a structured 5′ untranslated region (5′UTR) containing potential regulatory motifs like those in MCL-1 mRNA and the short half-life of MCL-1 protein. Pertinently, 2DG downregulated two other short-lived oncoproteins, MYC and MDM2. Thus, our results support MCL-1 as a critical 2DG target, but also reveal multiple effects on global translation that may well also affect its promotion of apoptosis.

Morphological evaluation of liposomal iron carriers.

Iron is one of the most important elements for human, because it plays an essential role in many metabolic processes. However, it is also recognized to be dangerous for its detrimental effect inside human cells, where, in the absence of homeostatic balance, it can induce free radicals formation. Moreover, an excessive accumulation of iron in tissues can produce iron overload, a condition incompatible with life. The use of liposomes as carriers can represent an interesting iron therapy to improve iron bioavailability and reduce its negative effects, in particular during pregnancy. In this study, a morphological analysis has been performed on commercial liposome vesicles at various drying times, both in saline solution and in distilled water. Furthermore, to highlight their possible interaction or internalization in cells, liposomes have been administered to human hemopoietic U937 cells. Ultrastructural analyses confirm that vesicle morphology and size are comparable with classical liposomal structures. Products are stable during specimen preparation and drying. Additionally, they have a good ability to penetrate into cells, interacting with cytoplasmic organelles, without inducing, at least apparently, any ultrastructural damage.

Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells.

The human hemopoietic progenitor hierarchy producing lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but remain incompletely characterized. Here, we demonstrated cord blood lympho-myeloid containing progenitor populations - the lymphoid-primed multi-potential progenitor (LMPP), granulocyte-macrophage progenitor (GMP) and multi-lymphoid progenitor (MLP) - were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Though most progenitors had uni-lineage myeloid or lymphoid potential, bi- and rarer multi-lineage progenitors occurred in LMPP, GMP and MLP. This, coupled with single cell expression analyses, suggested a continuum of progenitors execute lymphoid and myeloid differentiation rather than only uni-lineage progenitors being present downstream of stem cells.

Human Hemopoietic Progenitor Cell Toll-like and Thymic Stromal Lymphopoietin Receptor Expression and Function in Allergic Asthmatic Subjects

Human Hemopoietic Progenitor Cell Toll-like and Thymic Stromal Lymphopoietin Receptor Expression and Function in Allergic Asthmatic Subjects

Alpha-Particle-Induced Complex Chromosome Exchanges Transmitted through Extra-Thymic Lymphopoiesis In Vitro Show Evidence of Emerging Genomic Instability.

Human exposure to high-linear energy transfer α-particles includes environmental (e.g. radon gas and its decay progeny), medical (e.g. radiopharmaceuticals) and occupational (nuclear industry) sources. The associated health risks of α-particle exposure for lung cancer are well documented however the risk estimates for leukaemia remain uncertain. To further our understanding of α-particle effects in target cells for leukaemogenesis and also to seek general markers of individual exposure to α-particles, this study assessed the transmission of chromosomal damage initially-induced in human haemopoietic stem and progenitor cells after exposure to high-LET α-particles. Cells surviving exposure were differentiated into mature T-cells by extra-thymic T-cell differentiation in vitro. Multiplex fluorescence in situ hybridisation (M-FISH) analysis of naïve T-cell populations showed the occurrence of stable (clonal) complex chromosome aberrations consistent with those that are characteristically induced in spherical cells by the traversal of a single α-particle track. Additionally, complex chromosome exchanges were observed in the progeny of irradiated mature T-cell populations. In addition to this, newly arising de novo chromosome aberrations were detected in cells which possessed clonal markers of α-particle exposure and also in cells which did not show any evidence of previous exposure, suggesting ongoing genomic instability in these populations. Our findings support the usefulness and reliability of employing complex chromosome exchanges as indicators of past or ongoing exposure to high-LET radiation and demonstrate the potential applicability to evaluate health risks associated with α-particle exposure.

Potential of Herpesvirus Saimiri-Based Vectors To Reprogram a Somatic Ewing's Sarcoma Family Tumor Cell Line

Herpesvirus saimiri (HVS) infects a range of human cell types with high efficiency. Upon infection, the viral genome can persist as high-copy-number, circular, nonintegrated episomes that segregate to progeny cells upon division. This allows HVS-based vectors to stably transduce a dividing cell population and provide sustained transgene expression in vitro and in vivo. Moreover, the HVS episome is able to persist and provide prolonged transgene expression during in vitro differentiation of mouse and human hemopoietic progenitor cells. Together, these properties are advantageous for induced pluripotent stem cell (iPSC) technology, whereby stem cell-like cells are generated from adult somatic cells by exogenous expression of specific reprogramming factors. Here we assess the potential of HVS-based vectors for the generation of induced pluripotent cancer stem-like cells (iPCs). We demonstrate that HVS-based exogenous delivery of Oct4, Nanog, and Lin28 can reprogram the Ewing's sarcoma family tumor cell line A673 to produce stem cell-like colonies that can grow under feeder-free stem cell culture conditions. Further analysis of the HVS-derived putative iPCs showed some degree of reprogramming into a stem cell-like state. Specifically, the putative iPCs had a number of embryonic stem cell characteristics, staining positive for alkaline phosphatase and SSEA4, in addition to expressing elevated levels of pluripotent marker genes involved in proliferation and self-renewal. However, differentiation trials suggest that although the HVS-derived putative iPCs are capable of differentiation toward the ectodermal lineage, they do not exhibit pluripotency. Therefore, they are hereby termed induced multipotent cancer cells.

Differentiation of insulin-producing cells from human cord bloodderived haemopoietic stem cells in vitro

The use of stem cells in regenerative medicine holds great promise for the cure of many diseases, including type 1 diabetes mellitus, which, despite of the advances in current therapeutic approaches, remains to be one of the most serious health care problems. In addition to the traditional sources of adult stem cells, human umbilical cord blood has provided an important source of stem cells for research due to its unique advantages compared to other sources. In this study, we aimed at optimizing culture conditions for obtaining insulin-producing cells from cord blood hematopoietic stem cells. Twenty cord blood samples were subjected to short-term, liquid static culture that favors the proliferation of CD34+ hematopoietic stem cells. A duplicate culture was set for each sample, one with high glucose concentration and the other with low glucose concentration. Then these cells were subsequently induced to transdifferentiate into insulin-producing cells via a biphasic liquid culture using exendin-4. The expression of human insulin was then tested using RT-PCR. At the end of the culture, 17 out of the 20 samples (85%) cultured in high glucose concentration showed positive human insulin mRNA expression, while culture media with low glucose concentration failed to induce transdifferentiation into insulin-producing cells in any of the 20 samples. In brief, our study demonstrate that hematopoietic cord blood stem cells can transdifferentiate into insulin-producing cells in short-term liquid culture supplemented with high glucose concentration, nicotinamide, and exendin-4 in vitro.

Long-term Regulation of Genetically Modified Primary Hematopoietic Cells in Dogs

We report long-term results from a large animal model of in vivo selection. Nine years ago, we transplanted two dogs (E900 and E958) with autologous marrow CD34(+) cells that had been transduced with a gammaretrovirus vector encoding a conditionally activatable derivative of the thrombopoietin receptor. Receptor activation through administration of a chemical inducer of dimerization (CID) (AP20187 or AP1903) confers a growth advantage. We previously reported responses to two 30-day intravenous (i.v.) courses of AP20187 administered within the first 8 months post-transplantation. We now report responses to 5-day subcutaneous (s.c.) courses of AP20187 or AP1903 at months 14, 90, and 93 (E900), or month 18 (E958), after transplantation. Long-term monitoring showed no rise in transduced cells in the absence of drug. Retroviral insertion site analysis showed that 4 of 6 (E958) and 5 of 12 (E900) transduced hematopoietic cell clones persisted lifelong. Both dogs were euthanized for reasons unrelated to the gene therapy treatment at 8 years 11 months (E958) and 11 years 1 month (E900) of age. Three clones from E900 remained detectable in each of two secondary recipients, one of which was treated with, and responded to, AP1903. Our results demonstrate the feasibility of safely regulating genetically engineered hematopoietic cells over many years.

Expression of P2X Receptor Subtypes on CD34+ Cells and c-kit+ Cells of Human Umbilical Blood

The presence of several subtypes of P2X receptors on early hemopoietic precursors (CD34+) from human umbilical blood was detected by flow cytometry. The expression of P2X receptors on umbilical blood lymphocytes was an order of magnitude higher than that on adult human blood cells. Our results attest to early involvement of P2X receptors in differentiation of human hemopoietic cells.

LPA Induces Erythropoiesis Process Through Activating LPA Receptor 3

Lysophosphatidic acid (LPA), an extracellular lipid mediator, exerts multiple bioactivities through an activating family set of G-protein-coupled receptors (GPCRs). LPA receptor 3 (EDG7) is a member of endothelial differentiation gene (EDG) family receptors, which regulates in differentiation and development in circulation system. In zebrafish study using the zlpa3 anti-sense morpholino oligonucletids (zlpa3-Mo), hemopoietic defects was observed in the injected embryos. Cultured cord blood-derived human hemopoietic stem cells (hHSCs) could be induced to differentiate into mature erythrocytes in vitro by erythropoietin (EPO) and stem cell factor (SCF) stimulation. Erythroblasts express mRNA transcripts for both LPA1 and LPA3. Ki16425, a completive antagonist of LPA1 and LPA3, blocked the erythropoietic differentiation as detected through mRNA and protein expression of CD71 (Transferrin receptor protein 1) and GlyA (Glycophorin A). Furthermore, specifically knockdown LPA3, but not LPA1, blocked the erythropoiesis process. In addition, LPA enhanced the differentiation of erythroblasts in the plasma-free culture in a concentration dependant manner. By immunefluorescence analysis, translocation of β-catenin into nucleus, a downstream response of LPA receptor activation, was blocked by Ki16425 treatment. In conclusion, LPA participates in EPO dependant erythropoiesis process through activating LPA3, and β-catenin may be involved in this process.

Uncovering Haematopoietic System Dynamics and Single Multipotent Progenitors Activity In Vivo In Humans by Retroviral Tagging.

AbstractAbstract 2611The long-standing model of human haematopoiesis postulates that myeloid and lymphoid lineages branch separately at very early stages, producing myeloid or erythroid cells and T or B cells, respectively. Conversely, a revised scheme of haematopoietic hierarchy was recently proposed, in which myeloid cells represent a prototype of blood cells, while erythroid, T and B cells are specialized cell types. The validity of these models has been mainly tested in vivo in the mouse, and in vitro through clonal assays on human haemopoietic stem cells (HSC). However, a clear definitive elucidation of the real nature of human haemopoiesis should ideally involve the ability to track the dynamics, survival and differentiation potential of haemopoietic progenitor clones for a long period of time directly in vivo in humans. Upon retroviral gene transfer, transduced cells are univocally tagged by vector insertions allowing them to be distinguished and tracked in vivo by integration profiling. We previously showed that gene therapy (GT) for adenosine deaminase (ADA) deficient SCID based on infusion of transduced CD34+ cells and reduced intensity conditioning, resulted in full multilineage engraftment, in the absence of aberrant expansions. Therefore, long-term studies in these patients provide a unique human model to study in depth haemopoietic clonal dynamics by retroviral tagging. For this reason, we performed a comprehensive multilineage longitudinal insertion profile of bone marrow (BM) (CD34+, CD15+, CD19+, Glycophorin+) and peripheral blood (PB) (CD15+, CD19+, CD4+, CD8+ cells, naïve and memory T cell subpopulations) cells in 4 patients 3–6 years after GT, retrieving to date 1055 and 1999 insertions from BM and PB cell lineages respectively. We could shape the insertional landscape of each lineage through a tri-factorial analysis based on the number of integrations retrieved, the percentage of vector positive cells and the number of insertion shared with other lineages. We were able to uncover the effects of selective advantages of gene-corrected cells in periphery and the frequency of identical integrants in different haematopoietic compartments. BM cells displayed the highest proportion of shared integrants (up to 58.1%), reflecting the real-time repopulating activity of gene-corrected progenitors. On the other hand, PB samples carried in general a higher frequency of vector positive cells, with the exception of PB CD15+ cells showing insertional landscapes very similar to the one of BM lineages. Interestingly, the detection of exclusively shared myeloid-T\\B or myeloid-erythroid integrants may be supportive of a myeloid-based haemopoiesis model. We also uncovered “core integrants”, shared between CD34+ cells and both lymphoid and myeloid lineages, stably tagging active long-term multipotent progenitors overtime. Strikingly, one of these progenitor clones carried an insertion inside one of the two fragile sites of MLLT3 gene, involved by translocation events in mixed lineage leukemia. We were able to track this and another integrant (downstream the LRRC30 gene) by specific PCRs, confirming the multilineage contribution to haematopoiesis of the relative progenitor clones and their fluctuating lineage outputs over 4 years, without showing aberrant expansions. We also retrieved 170 and 174 integrations from 4 T cell subtypes (Naive, TEMRA, Central and Effector memory) in two patients under PBL-GT and HSC-GT respectively. We found evidences that single naive T cell clones may survive in patients for up to 10 years after last infusion while maintaining their differentiation capacity into different T cell subpopulations. Interestingly, a cluster of 4 insertions (one of them shared among all T cell subtypes) was found in proximity of the interferon regulatory factor 2 binding protein 2 (IRF2BP2) gene in naive T cells from PBL-GT patient, thus suggesting an influence of transcriptional activity of this region on selective advantage of gene-corrected lymphocytes. In conclusion, through retroviral tagging, we can uniquely track single transduced haemopoietic cells directly in vivo in humans. The application of mathematical models to our insertion datasets is allowing to uncover new information on the fate and activity of haematopoietic progenitors and their differentiated progeny years after transplantation in GT patients. Disclosures:No relevant conflicts of interest to declare.

Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells

Surprisingly little is known about the effects of the physical microenvironment on hemopoietic stem and progenitor cells. To explore the physical effects of matrix elasticity on well-characterized primitive hemopoietic cells, we made use of a uniquely elastic biomaterial, tropoelastin. Culturing mouse or human hemopoietic cells on a tropoelastin substrate led to a two- to threefold expansion of undifferentiated cells, including progenitors and mouse stem cells. Treatment with cytokines in the presence of tropoelastin had an additive effect on this expansion. These biological effects required substrate elasticity, as neither truncated nor cross-linked tropoelastin reproduced the phenomenon, and inhibition of mechanotransduction abrogated the effects. Our data suggest that substrate elasticity and tensegrity are important mechanisms influencing hemopoietic stem and progenitor cell subsets and could be exploited to facilitate cell culture.

Identification and biophysical assessment of the molecular recognition mechanisms between the human haemopoietic cell kinase Src homology domain 3 and ALG-2-interacting protein X

SFKs (Src family kinases) are central regulators of many signalling pathways. Their functions are tightly regulated through SH (Src homology) domain-mediated protein-protein interactions. A yeast two-hybrid screen using SH3 domains as bait identified Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] as a novel Hck (haemopoietic cell kinase) SH3 domain interactor. The Alix-Hck-SH3 interaction was confirmed in vitro by a GST (glutathione transferase) pull-down assay and in intact cells by a mammalian two-hybrid assay. Furthermore, the interaction was demonstrated to be biologically relevant in cells. Through biophysical experiments, we then identified the PRR (proline-rich region) motif of Alix that binds Hck-SH3 and determined a dissociation constant of 34.5 μM. Heteronuclear NMR spectroscopy experiments were used to map the Hck-SH3 residues that interact with an ALIX construct containing the V and PRR domains or with the minimum identified interacting motif. Finally, SAXS (small-angle X-ray scattering) analysis showed that the N-terminal PRR of Alix is unfolded, at least before Hck-SH3 recognition. These results indicate that residues outside the canonical PxxP motif of Alix enhance its affinity and selectivity towards Hck-SH3. The structural framework of the Hck-Alix interaction will help to clarify how Hck and Alix assist during virus budding and cell-surface receptor regulation.

Towards cellular therapy with a spring in your step!

Surprisingly little is known regarding the effects of the physical microenvironmental niche on haemopoietic stem cells. We have explored the effects of matrix elasticity on stem cell properties using a unique synthetic substrate, tropoelastin, which we used to show that both murine and human primitive haemopoietic cells are more efficiently maintained on an elastically extensible substrate than controls. We have shown that culturing primitive murine and human cord blood haemopoietic cells on tropoelastin in vitro led to increases in cell number, clonogenicity, proliferation and engraftment following transplantation. Atomic force measurements of truncations and crosslinked tropoelastin strengthen the hypothesis that stem cells sense the elasticity of their microenvironment. A consequence of this idea is that alterations to niche elasticity in disease states may contribute to abnormal haemopoiesis. Further, elastic substrates such as tropoelastin may offer a new approach to biomaterial design aimed to achieve optimal ex vivo culture conditions for primitive haemopoietic cells. In order to take advantage of this type of observation arising from the basic research laboratory (and many other opportunities in cellular therapies), a basic introduction in the principles and regulatory aspects of good manufacturing practice of cellular therapeutics will be provided.

Thrombopoietic‐mesenchymal interaction that may facilitate both endochondral ossification and platelet maturation via CCN2

CCN2 plays a central role in the development and growth of mesenchymal tissue and promotes the regeneration of bone and cartilage in vivo. Of note, abundant CCN2 is contained in platelets, which is thought to play an important role in the tissue regeneration process. In this study, we initially pursued the possible origin of the CCN2 in platelets. First, we examined if the CCN2 in platelets was produced by megakaryocyte progenitors during differentiation. Unexpectedly, neither megakaryocytic CMK cells nor megakaryocytes that had differentiated from human haemopoietic stem cells in culture showed any detectable CCN2 gene expression or protein production. Together with the fact that no appreciable CCN2 was detected in megakaryocytes in vivo, these results suggest that megakaryocytes themselves do not produce CCN2. Next, we suspected that mesenchymal cells situated around megakaryocytes in the bone marrow were stimulated by the latter to produce CCN2, which was then taken up by platelets. To evaluate this hypothesis, we cultured human chondrocytic HCS-2/8 cells with medium conditioned by differentiating megakaryocyte cultures, and then monitored the production of CCN2 by the cells. As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium. We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro. These findings indicate that megakaryocytes secrete some unknown soluble factor(s) during differentiation, which factor stimulates the mesenchymal cells to produce CCN2 for uptake by the platelets. We also consider that, during bone growth, such thrombopoietic-mesenchymal interaction may contribute to the hypertrophic chondrocyte-specific accumulation of CCN2 that conducts endochondral ossification.

Gravitational field-flow fractionation of human hemopoietic stem cells

New cell sorting methodologies, which are simple, fast, non-invasive, and able to isolate homogeneous cell populations, are needed for applications ranging from gene expression analysis to cell-based therapy. In particular, in the forefront of stem cell isolation, progenitor cells have to be separated under mild experimental conditions from complex heterogeneous mixtures prepared from human tissues. Most of the methodologies now employed make use of immunological markers. However, it is widely acknowledged that specific markers for pluripotent stem cells are not as yet available, and cell labelling may interfere with the differentiation process. This work presents for the first time gravitational field-flow fractionation (GrFFF), as a tool for tag-less, direct selection of human hematopoietic stem and progenitor cells from cell samples obtained by peripheral blood aphaeresis. These cells are responsible to repopulate the hemopoietic system and they are used in transplantation therapies. Blood aphaeresis sample were injected into a GrFFF system and collected fractions were characterized by flow cytometry for CD34 and CD45 expression, and then tested for viability and multi-differentiation potential. The developed GrFFF method allowed obtaining high enrichment levels of viable, multi-potent hematopoietic stem cells in specific fraction and it showed to fulfil major requirements of analytical performance, such as selectivity and reproducibility of the fractionation process and high sample recovery.

Specific Interaction of Cultured Human Mesenchymal and Hemopoietic Stem Cells under Conditions of Reduced Oxygen Content

We studied the effect of reduced oxygen content (5%) on the phenotype and functional activity of cultured human mesenchymal stem cells. The expression of main immunophenotypic markers for mesenchymal stem cells (CD13, CD29, CD44, CD73, CD90, CD105, and HLA-I) remained practically unchanged under conditions of hypoxia. The expression of cell adhesion molecules (CD54 and CD106) increased during coculturing of mesenchymal stem cells and hemopoietic stem cells. These changes were accompanied by increased production of hemopoietins (interleukin-6 and interleukin-8) and enhanced colony-forming capacity of hemopoietic stem cells. Coculturing of mesenchymal stem cells and hemopoietic stem cells during hypoxia was followed by increased formation of hemopoietic islets and intensive production of interleukin-6, interleukin-8, and vascular endothelial growth factor (compared to cultures under normoxic conditions).