The peripheral chimerism of bone marrow–derived stem cells after transplantation: regeneration of gastrointestinal tissues in lethally irradiated mice

Bone marrow–derived cells represent a heterogeneous cell population containing haematopoietic stem and progenitor cells. These cells have been identified as potential candidates for use in cell therapy for the regeneration of damaged tissues caused by trauma, degenerative diseases, ischaemia and inflammation or cancer treatment. In our study, we examined a model using whole-body irradiation and the transplantation of bone marrow (BM) or haematopoietic stem cells (HSCs) to study the repair of haematopoiesis, extramedullary haematopoiesis and the migration of green fluorescent protein (GFP⁺) transplanted cells into non-haematopoietic tissues. We investigated the repair of damage to the BM, peripheral blood, spleen and thymus and assessed the ability of this treatment to induce the entry of BM cells or GFP⁺lin⁻Sca-1⁺ cells into non-haematopoietic tissues. The transplantation of BM cells or GFP⁺lin⁻Sca-1⁺ cells from GFP transgenic mice successfully repopulated haematopoiesis and the haematopoietic niche in haematopoietic tissues, specifically the BM, spleen and thymus. The transplanted GFP⁺ cells also entered the gastrointestinal tract (GIT) following whole-body irradiation. Our results demonstrate that whole-body irradiation does not significantly alter the integrity of tissues such as those in the small intestine and liver. Whole-body irradiation also induced myeloablation and chimerism in tissues, and induced the entry of transplanted cells into the small intestine and liver. This result demonstrates that grafted BM cells or GFP⁺lin⁻Sca-1⁺ cells are not transient in the GIT. Thus, these transplanted cells could be used for the long-term treatment of various pathologies or as a one-time treatment option if myeloablation-induced chimerism alone is not sufficient to induce the entry of transplanted cells into non-haematopoietic tissues.     

Vascular Calcifying Progenitor Cells Possess Bidirectional Differentiation Potentials

Vascular calcification is an advanced feature of atherosclerosis for which no effective therapy is available. To investigate the modulation or reversal of calcification, we identified calcifying progenitor cells and investigated their calcifying/decalcifying potentials. Cells from the aortas of mice were sorted into four groups using Sca-1 and PDGFRα markers. Sca-1⁺ (Sca-1⁺/PDGFRα⁺ and Sca-1⁺/PDGFRα⁻) progenitor cells exhibited greater osteoblastic differentiation potentials than Sca-1⁻ (Sca-1⁻/PDGFRα⁺ and Sca-1⁻/PDGFRα⁻) progenitor cells. Among Sca-1⁺ progenitor populations, Sca-1⁺/PDGFRα⁻ cells possessed bidirectional differentiation potentials towards both osteoblastic and osteoclastic lineages, whereas Sca-1⁺/PDGFRα⁺ cells differentiated into an osteoblastic lineage unidirectionally. When treated with a peroxisome proliferator activated receptor γ (PPARγ) agonist, Sca-1⁺/PDGFRα⁻ cells preferentially differentiated into osteoclast-like cells. Sca-1⁺ progenitor cells in the artery originated from the bone marrow (BM) and could be clonally expanded. Vessel-resident BM-derived Sca-1⁺ calcifying progenitor cells displayed nonhematopoietic, mesenchymal characteristics. To evaluate the modulation of in vivo calcification, we established models of ectopic and atherosclerotic calcification. Computed tomography indicated that Sca-1⁺ progenitor cells increased the volume and calcium scores of ectopic calcification. However, Sca-1⁺/PDGFRα⁻ cells treated with a PPARγ agonist decreased bone formation 2-fold compared with untreated cells. Systemic infusion of Sca-1⁺/PDGFRα⁻ cells into Apoe^−/− mice increased the severity of calcified atherosclerotic plaques. However, Sca-1⁺/PDGFRα⁻ cells in which PPARγ was activated displayed markedly decreased plaque severity. Immunofluorescent staining indicated that Sca-1⁺/PDGFRα⁻ cells mainly expressed osteocalcin; however, activation of PPARγ triggered receptor activator for nuclear factor-κB (RANK) expression, indicating their bidirectional fate in vivo. These findings suggest that a subtype of BM-derived and vessel-resident progenitor cells offer a therapeutic target for the prevention of vascular calcification and that PPARγ activation may be an option to reverse calcification.     

The isolation and in vitro expansion of hepatic Sca-1 progenitor cells

The intra-hepatic population of liver progenitor cells expands during liver injury when hepatocyte proliferation is inhibited. These cells can be purified by density gradient centrifugation and cultured. Separated by size only this population contains small cells of hematopoietic, epithelial and endothelial lineages and is thought to contain liver stem cells. The identity of liver stem cells remains unknown although there is some evidence that tissue Sca1⁺ CD45⁻ cells display progenitor cell characteristics. We identified both intra-hepatic and gall bladder Sca1⁺ cells following liver injury and expanded ex vivo Sca1 cells as part of heterogenous cell culture or as a purified population. We found significant difference between the proliferation of Sca-1 cells when plated on laminin or collagen I while proliferation of heterogenous population was not affected by the extracellular matrix indicating the necessity for culture of Sca1⁺ cells with laminin matrix or laminin producing cells in long term liver progenitor cell cultures.     

Persistent activation of omentum influences the pattern of muscular lesion in the mdx diaphragm

The mdx (X chromosome-linked muscular dystrophy) mouse develops a multi-staged disorder characterized by muscle degeneration and reactive fibrosis. Skeletal muscles of mdx mice are not equally susceptible to degeneration. The aim of this study was to verify whether the intense remodeling of the mdx diaphragm could be attributed to influences from the peritoneal microenvironment and omentum, a lymphohematopoietic tissue rich in progenitor cells and trophic factors. At ages corresponding to increased muscular regeneration (12?weeks) and activation of fibrosis (24?weeks), the mdx omentum exhibited (1) morphological and functional characteristics of activation with enlarged milk-spots, an accumulation of CD4⁺, CD8⁺ and CD19⁺B220⁺ B lymphocytes; (2) the formation of clusters positive for proliferating cell nuclear antigen, mainly in B220⁺-rich areas organized in a follicular structure with a germinative center without any challenge by external antigen inducers; (3) clusters with cells positive for fibroblast growth factor-2, numerous Sca-1⁺CD3^-CD19^-Mac-1^- progenitor cells and increased CD4⁺, CD8⁺ and CD3⁺NK1.1⁺ cells in the peritoneal cavity. Omentectomy reduced areas with F4/80⁺ inflammatory infiltrate the activity of matrix metalloproteases 9 and 2, collagen deposition and areas with regenerating myofibers in the diaphragm. Thus, persistent activation of the omentum influences the pattern of inflammation and regeneration of the mdx diaphragm partly via the activation of progenitor cells and the production of growth factors that influence the physiopathology of the muscular tissue remodeling.     

Immunohistological Localization of Endogenous Unlabeled Stem Cells in Wounded Skin

Various types of endogenous stem cells (SCs) participate in wound healing in the skin at different anatomical locations. SCs need to be identified through multiple markers, and this is usually performed using flow cytometry. However, immunohistological identification of endogenous stem cells in the skin at different anatomical locations by co-staining multiple SC markers has been seldom explored. We examined the immunohistological localization of four major types of SCs in wounded skin by co-staining for their multiple markers. Hematopoietic SCs were co-stained for Sca1 and CD45; mesenchymal SCs for Sca1, CD29, and CD106; adipose SCs for CD34, CD90, and CD105; and endothelial progenitor cells and their differentiated counterparts were co-stained for CD34, Tie2, and von Willebrand factor. We found Sca1⁺CD45⁺ SCs in the epidermis, dermis and hypodermis of wounded skin. Sca1⁺CD29⁺ and Sca1⁺CD106⁺ mesenchymal SCs, CD34⁺CD105⁺, CD34⁺CD90⁺, and CD90⁺CD105⁺ adipose SCs, as well as CD34⁺Tie2⁺ endothelial progenitor cells were also located in the epidermis, dermis, and hypodermis. This study demonstrates the feasibility of using immunohistological staining to determine the location of SCs in wounded skin and the intracellular distribution of their molecular markers.     

Testosterone influences renal electrolyte excretion in SHR/y and WKY males

Background

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH⁺ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH⁺ cellular subsets in bone marrow from pure red cell aplasia patients.

Results

In normal bone marrow, we identified three populations of cells, namely ALDH⁺CD34⁺, ALDH^-CD34⁺ and ALDH⁺CD34^- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH^-CD34⁺ cells, ALDH⁺CD34⁺ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH⁺CD34^- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH⁺CD34^- cells showed a CD71^bright, CD105⁺, CD45^- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH⁺CD34^- precursors were not detectable in patients with pure red cell aplasia (PRCA).

Conclusion

Our study, comparing surface antigen expression of ALDH⁺/CD34⁺, ALDH^-/CD34⁺ and ALDH⁺/CD34^- progenitor cell subsets in human bone marrow, clearly indicated that ALDH⁺CD34^- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.     

Expression of dual Nucleotides/Cysteinyl‐Leukotrienes Receptor GPR17 in early trafficking of cardiac stromal cells after myocardial infarction

GPR17 is a G_i-coupled dual receptor activated by uracil-nucleotides and cysteinyl-leukotrienes. These mediators are massively released into hypoxic tissues. In the normal heart, GPR17 expression has been reported. By contrast, its role in myocardial ischaemia has not yet been assessed. In the present report, the expression of GPR17 was investigated in mice before and at early stages after myocardial infarction by using immunofluorescence, flow cytometry and RT-PCR. Before induction of ischaemia, results indicated the presence of the receptor in a population of stromal cells expressing the stem-cell antigen-1 (Sca-1). At early stages after ligation of the coronary artery, the receptor was expressed in Sca-1⁺ cells, and cells stained with Isolectin-B4 and anti-CD45 antibody. GPR17⁺ cells also expressed mesenchymal marker CD44. GPR17 function was investigated in vitro in a Sca-1⁺/CD31⁻ cell line derived from normal hearts. These experiments showed a migratory function of the receptor by treatment with UDP-glucose and leukotriene LTD4, two GPR17 pharmacological agonists. The GPR17 function was finally assessed in vivo by treating infarcted mice with Cangrelor, a pharmacological receptor antagonist, which, at least in part, inhibited early recruitment of GPR17⁺ and CD45⁺ cells. These findings suggest a regulation of heart-resident mesenchymal cells and blood-borne cellular species recruitment following myocardial infarction, orchestrated by GPR17.     

Immunophenotype and differentiation capacity of bone marrow-derived mesenchymal stem cells from CBA/Ca,ICR and Balb/c mice

AIM:To assess the capacity to isolate and expand mesenchymal stem cells(MSC)from bone marrow of CBA/Ca,ICR and Balb/c mice. METHODS:Bone marrow of tibia and femur were flushed,cultured and maintained in supplemented Dulbecco’s modified Eagle’s medium.MSC immunophenotype of cultures were tracked along increasing passages for positivity to CD106,Sca-1 and CD44 and negativity to CD45,CD11b and MHC classⅡ.Differentiation capacity of MSC towards osteogenic and adipo-genic lineages were also assessed. RESULTS:MSC were successfully cultured from bone marrow of all 3 strains,albeit differences in the temporal expression of certain surface antigens.Their differentiation into osteocytes and adipocytes were also observed. MSC from all 3 mouse strains demonstrated a shift from a haematopoietic phenotype(CD106-CD45+CD11b+Sca-1low)to typical MSC phenotype(CD106+CD45-CD11b-Sca-1high)with increasing passages. CONCLUSION:Information garnered assists us in the decision of selecting a mouse strain to generate MSC from for downstream experimentation.     

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time¹. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).Both trabecular osteoblasts^2,3 and sinusoidal endothelium⁴ constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin⁺ osteoblasts³ and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-1⁵ concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity⁶. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands⁷ and interferon-α⁶ can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin^- c-Kit⁺ Sca-1⁺ CD150⁺ CD48^- CD34^- population has been described⁸. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs⁹. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described¹⁰. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.     

Sca-1+ cardiosphere-derived cells are enriched for Isl1-expressing cardiac precursors and improve cardiac function after myocardial injury

Background

Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.

Methodology/Principal Finding

Using “middle aged” mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1⁺CD45^- cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1⁺CD45^- cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1⁺CD45^- cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts.

Conclusions/Significance

These studies demonstrate that cloned Sca-1⁺CD45^- cells derived from CSs from infarcted “middle aged” hearts are enriched for second heart field (i.e., Isl-1⁺) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.     

Isolation murine mesenchymal stem cells by positive selection

Isolation and purification of mesenchymal stem cells (MSCs) from mouse via plastic adherent cultures is arduous because of the unwanted growth of hematopoietic cells and non-MSCs. In this work, homogenous populations of CD34⁺ MSCs from mouse bone marrow were isolated via positive selection. For this purpose, C57Bl/6 mice were killed and bone marrow cells were aspirated before incubation with magnetic bead conjugated to anti-CD34 antibody. A sample of positively selected CD34⁺ cells were prepared for flow cytometry to examine the expression of CD34 antigen and others were subcultured in a 25-cm² culture flask. To investigate the mesenchymal nature, the plastic adherent cultivated cells were induced to differentiate along osteoblastic and adipogenic lineages. Furthermore, the expression of some surface markers was investigated by flow cytometry. According to the result, purified populations of fibroblast-like CD34⁺ cells were achieved in the first passage (1 wk after culture initiation). The cells expressed CD34, CD44, Sca-1, and Vcam-1 antigens (markers) but not CD11b and CD45. They were capable of differentiating into osteocytes and adipocytes. This study indicated that our protocol can result in the efficient isolation of homogenous populations of MSCs from C57BL/6 mouse bone marrow. We have shown that murine bone marrow-derived CD34⁺ cells with plastic adherent properties and capability of differentiating into skeletal lineages in vitro are MSCs.     

Characterization of human embryonic stem cell-derived hematopoietic progenitor phenotype

Differentiation of human embryonic stem cells (hESCs) into hematopoietic lineages using various methods has been reported. However, the phenotype that precisely defines the hematopoietic progenitor compartment with clonogenic activities has yet to be determined. Here, we measured and characterized progenitor function of subfractions of cells prospectively isolated from human embryoid bodies (hEBs) during hematopoietic differentiation basing on surface markers CD45, CD34, CD43, and CD38. We report that hematopoietic progenitors predominantly resided in the CD45⁺ subset. CD43⁺ cells lacking CD45 expression were largely devoid of progenitor activity. However, progenitor activity and multipotentiality was more enriched in CD45⁺ cells co-expressing CD43. CD45⁺ subset co-expressing CD34 but lacking CD38 expression (CD45⁺CD34⁺CD38^-) were further enriched for CFU capacity compared to the CD45⁺CD34⁺CD38⁺ subset. Our study demonstrates a role of CD43 in enriching hematopoietic progenitors derived from hEBs and reveals a hierarchical organization of hESC-derived hematopoietic progenitor compartments defined by phenotypic markers.     

Irradiated stem cells and ageing of the haematopoietic system

In the work presented here, changes in haematopoiesis of mice (B6129SF2/J) were studied 1 year after their whole-body exposure to a dose of 7 Gy (72% of mice survived). The irradiated mice were compared with non-irradiated younger (4 months of age) and older (16 months of age) mice. There was a significant increase in the relative abundance of primitive stem cells with long-term capability of the haematopoiesis recovery lin⁻/Sca-1⁺/CD117⁺/CD34⁻ in the bone marrow of mice aged 16 months (irradiated and non-irradiated) compared with those aged 4 months. In terms of the ability to respond to further whole-body irradiation at a dose of 1 Gy, the presence of γH2A.X foci was studied in lin⁻ bone marrow cells. There was a considerable number of persisting foci in lin⁻ stem cells isolated from the bone marrow of the older irradiated mice. In the blood count from the peripheral blood of the older mice (both non-irradiated and irradiated at 7 Gy), there was a significant increase in granulocytes. In the group exposed to 7 Gy, the numbers of thrombocytes significantly increased, and on the contrary, the numbers of erythrocytes, the amount of haemoglobin, and haematocrit significantly decreased.     

CD34+VEGFR‐3+ progenitor cells have a potential to differentiate towards lymphatic endothelial cells

Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization. However, it is poorly understood whether EPCs contribute to lymphangiogenesis. Here, we assessed differentiation of a novel population of EPCs towards lymphatic endothelial cells and their lymphatic formation. CD34⁺VEGFR‐3⁺ EPCs were isolated from mononuclear cells of human cord blood by fluorescence‐activated cell sorting. These cells expressed CD133 and displayed the phenotype of the endothelial cells. Cell colonies appeared at 7–10 days after incubation. The cells of the colonies grew rapidly and could be repeatedly subcultured. After induction with VEGF‐C for 2 weeks, CD34⁺VEGFR‐3⁺ EPCs could differentiate into lymphatic endothelial cells expressing specific markers 5′‐nucleotidase, LYVE‐1 and Prox‐1. The cells also expressed hyaluronan receptor CD44. The differentiated cells had properties of proliferation, migration and formation of lymphatic capillary‐like structures in three‐dimensional collagen gel and Matrigel. VEGF‐C enhanced VEGFR‐3 mRNA expression. After interfering with VEGFR‐3 siRNA, the effects of VEGF‐C were diminished. These results demonstrate that there is a population of CD34⁺VEGFR‐3⁺ EPCs with lymphatic potential in human cord blood. VEGF‐C/VEGFR‐3 signalling pathway mediates differentiation of CD34⁺VEGFR‐3⁺ EPCs towards lymphatic endothelial cells and lymphangiogenesis. Cord blood‐derived CD34⁺VEGFR‐3⁺ EPCs may be a reliable source in transplantation therapy for lymphatic regenerative diseases.     

Isolation of progenitor cells from cord blood using adhesion matrices

The aim of this study was to develop optimal conditions for selective adhesion and isolation of mesenchymal progenitor cells (MPCs) from cord blood and to determine their potential for osteogenic differentiation. Mononuclear cells (MNCs) were isolated by Ficoll-Paque gradient and plated onto 48-well culture plates precoated with: human or bovine collagen type I, human collagen type IV, fibronectin or matrigel. Cultures were incubated in αMEM containing fetal calf serum. Viability of the adherent cells was determined by alamarBlue® assay after 2, 3, and 4 weeks. After 4 weeks in culture, cells were typsinized and replated. Primary cultures were analyzed by histochemistry and third passage cells by FACS. Isolated fibroblast-like cells were cultured in the presence of osteogenic factors and differentiation determined by Alizarin Red S staining, RT-PCR and electron dispersive spectroscopy (EDS). MNCs adhered to all types of matrices with the greatest adhesion rates on fibronectin. These cells were CD45⁺, CD105⁺, CD14⁺, CD49a⁺, CD49f⁺, CD44⁺ and CD34⁻. The highest incidence of progenitor cells (PC) was observed on fibronectin and polystyrene. Passages were CD45⁻, CD14⁻, CD34⁻ and weakly CD105⁺. Primary cultures expressed endothelial/macrophage RNA markers whether cultured on fibronectin or polystyrene and these markers decreased upon passage. The best osteogenic differentiation was observed in MPCs cultured in osteogenic medium containing vitamin D₃ and FGF9. These cells expressed the bone-related mRNA, collagen type I, core binding factor I (Cbfa I), osteocalcin and osteopontin. EDS of deposits produced by these cells demonstrated a calcium/phosphate ratio parallel to hydroxyapatite. It was concluded that fibronectin increased adhesion rates and isolation potential of cord blood mesenchymal progenitor cells.     

Placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells support hematopoietic recovery of X-irradiated human CD34+ cells

AimsThe potential of human mesenchymal stem cell-like stroma prepared from placental/umbilical cord blood for hematopoietic regeneration by X-irradiated hematopoietic stem cells is herein assessed.Main methodsPlacental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells were applied to a regenerative ex vivo expansion of X-irradiated human CD34⁺ cells in a serum-free liquid culture supplemented with a combination of interleukine-3 plus stem cell factor plus thrombopoietin.Key findingsThe total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture. In addition, the number of CD34⁺ cells and CD34⁺/CD38^? cells, immature hematopoietic stem/progenitor cells also increased more than the stroma-free culture. The stromal cells produced various types of cytokines, although there was little difference between the co-cultures of non-irradiated and X-irradiated cells with stromal cells. Furthermore, when X-irradiated cells came in contact with stromal cells for 16 h before cytokine stimulation, a similar degree of hematopoiesis was observed, thus suggesting the critical role of cell-to-cell interaction.SignificanceThe present results showed the potential efficacy of human mesenchymal stem cell-like stroma for hematopoietic regeneration from irradiated hematopoietic stem/progenitor cells.     

Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro

Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56⁺ cells grew rapidly, a population of CD15⁺ cells emerged, partly from CD56⁺ cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56⁺ and CD15⁺ cells shared osteogenic and chondrogenic abilities, while CD56⁺ cells presented a myogenic capacity and CD15⁺ cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.     

Expression of early hematopoietic markers in cord and mobilized blood

G-CSF mobilized peripheral blood and cord blood are major sources of hematopoietic progenitor cells. These cells are characterized by expression of “early” antigens. We have evaluated the coexpression of hematopoietic markers CD34, CD133, CD90, CDCP1, and CD117 and activation antigen CD38 using multicolor flow cytometry. We showed that cells positive for each particular antigen generate a separate population. The percentage of cells expressing each particular “early” antigen is twice as high in the cord blood as in the mobilized blood. The cell number with complex progenitor phenotype (CD34⁺/CD38^?/CD117^?, CD133⁺/CD34⁺/CD38^?, CDCP1⁺/CD34⁺/CD38^?, etc.) is equal in mobilized and cord blood. There is a strong positive correlation between CD34, CD133, CD117, and CDCP1 expression in both groups. Positive correlation was observed between CD90 and CD34, CD133, CDCP1, and CD117 expression only in cord blood; it was not significant in mobilized blood. The analyses of early antigens’ coexpression with activation CD38 marker did not confirm the hypothesis of sequential activation and loss of expression of the aforementioned antigens. We assume that there is global regulation of CD34, CD133, CDCP1, and CD117 expression. Expression of CD38 may be reversibly suppressed during maturation of the hematopoietic cells, and CD117 may be expressed on not only myeloid cells.     

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer''s patch (PP). Common dendritic cell progenitors (CDPs, lin^- c-kit^lo CD115⁺ Flt3⁺) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar^-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1⁺ and recipients being CD45.2⁺. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar^-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.