Angiographic demonstration of neoangiogenesis after

Copyright © 2012 Cognizant Communication Corporation DOI: 10.3727/096368912X636993 CT-0580 Accepted 11/30/2011 for publication in “Cell Transplantation”

Full title: An Ectopic stromal implant model for hematopoietic reconstitution and in vivo evaluation of bone marrow niches Authors: Flávio Henrique Paraguassú-Braga1 , Ana Paula G. Alves2, Isabella Maria Alvim Andrade Santos

2,*

Martin Bonamino

2

and Adriana Bonomo

2,,3

1)Banco de Sangue de Cordão Umbilical e Placentário, Centro de Transplante de Medula Óssea, Instituto Nacional de Câncer, 2)Programa de Medicina Experimental, Coordenação Geral Técnico-Científica, Instituto Nacional de Câncer, 3)Departamento de Imunologia, Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro * IMAAS was supported with a Ministry of Health/INCA fellowship Short title: stroma implant model

Keywords: hematopoiesis, stroma, bone marrow, Cx43 Corresponding author: Adriana Bonomo Programa de Medicina Experimental Instituto Nacional de Câncer CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 1

Copyright © 2012 Cognizant Communication Corporation Rua André Cavalcanti, 37 - Centro 20231-050 - Rio de Janeiro - RJ Phone: +55 21 3207-6598; Fax: +55 21 3207 6509 Email: [email protected]

CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 2

Copyright © 2012 Cognizant Communication Corporation Abstract In adults, hematopoiesis takes places in the bone marrow, where specialized niches containing mesenchymal non-hematopoietic cells (stroma) harbor the Hematopoietic Stem Cell (HSC). These niches are responsible and essential for the maintenance of HSCs. Attempts to expand HSCs fail to keep the general properties of stem cells which depend on several niche components difficult to reproduce in in vitro culture systems.

Here, we describe a methodology for in vivo study of hematopoietic

stroma. We use stroma loaded macroporous microcarriers implanted in the subcutaneous tissue of experimental animals and show that the Ectopic Stroma Implant (ESI) is able to support hematopoiesis. Moreover, lethally irradiated mice can be rescued by ESI pre-loaded with HSCs, showing that they function as an ectopic bone marrow. ESI is also shown as a good system to study the role of different niche components. As an example we used stromas lacking connexin 43 (Cx43) and confirm the importance of this molecule in the maintenance of the HSC niche in vivo. We believe ESI can work as an ectopic bone marrow allowing in vivo testing of different niches components and opening new avenues for the treatment of a variety of hematologic conditions particularly when stromal cell defects are the main cause of disease.


Copyright © 2012 Cognizant Communication Corporation Introduction Hematopoietic Stem Cells (HSC) are characterized by their self renewal capacity and ability to differentiate into different hematopoietic progenitor cells (HPCs), giving rise to all blood lineages (57) . In adulthood hematopoiesis takes places inside the bones of the axial skeleton where specialized niches containing mesenchymal and other hematopoietic and non-hematopoietic cells (generally named as stroma) harbor the HSCs. These niches are responsible and essential for maintenance and development of the HSCs (1,55). Alterations in bone marrow originate a number of pathologies, as malignancies and aplasias. This can be caused by defects in hematopoietic cells or stroma components. Defects in hematopoietic cells are frequently associated with malignancies. Stromal defects are believed to confer pathological alterations in hematopoiesis as well, as has been shown in myelodisplastic syndromes (7) and aplastic anemia (11). Hematopoietic stem cell reconstitution is widely used to treat a great number of hematologic diseases. However this procedure is hampered by the occurrence of graft versus host disease (GVHD) – a severe disease with high morbidity and mortality rates (6,9). Transfer of highly purified CD34+ cells can overcome GVHD in cases where immune responses against malignant cells are not mandatory (3,14). Enrichment in immature progenitors also enables the use umbilical cord blood (UCB) as transplant product to be used in adults. Transplants using UCB are advantageous over bone marrow or peripheral blood as they are readily available and allow MHC disparity with comparable lower incidence of GVHD (52). CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 4

Copyright © 2012 Cognizant Communication Corporation However, generation or expansion of high numbers of immature hematopoietic progenitors in vitro has been limited by the complex array interactions present in hematopoietic niches. When cultured in vitro, HSCs quickly lose their characteristics (25,59). Growth factors, hormones, extracellular matrix proteins and intercellular interactions has been shown critical for maintenance of long term hematopoietic stem cells, enlightening the difficulties to reproduce the niche milieu in vitro

(24,31,32,46,49-51,54) Three dimensional cultures has also been explored as a way to expand and study HSCs (4,5,23,44,45) but even though achievements are far from the actual in vivo expansion capacity of HSCs (46). Hematopoietic stem cell reconstitution gains an even higher level of complexity when the defect is in the stroma, because stroma transplants usually do not seed the bone marrow (29) although in aplastic anemia (21) allogeneic mesenchymal stem cell engraftment had been reported to correct stromal defects (16). Interesting, in some systems it has been shown to improve HSC engraftment (35) but the mechanism apparently lie in the immunosuppressive effect. Moreover, since mesenchymal cells are highly undifferentiated and can give rise to a number of other tissues, caution must be taken on its therapeutic use (29,51). One alternative to correct hematopoietic failures rely on the generation of Bone Marrow Organ Systems (BMOS) able to ectopically reproduce a functional bone marrow. In fact, grafts using hydroxyapatite tricalcium phosphate (HA/TCP) support has first been reported as osteogenic support to allow bone formation in vivo (26). More recently, HA loaded mesenchymal stem cell was shown to give rise to BMOS, CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 5

Copyright © 2012 Cognizant Communication Corporation characterized by trabecular bone and hematopoietic tissue development which could rescue lethally irradiated experimental animals (36). This same model system was used in ageing studies to show that transplanted mesenchymal cells act in distant sites delaying age related phenomena (58). Here we present an alternative method to generate BMOS using macroporous microcarriers commonly used in tissue culture. Unlike the use of HA/TCP carriers, which were developed towards bone reconstitution (26), cellulose microcarriers were originally designed to increase tissue culture surface area. In vivo, we used them as a 3D model of stroma implant (Ectopic Stroma Implant – ESI) which supports hematopoiesis. Cellulose microcarriers present two advantages over the HA/TCPA as vehicles for stromal cells: they are rapidly absorbed when empty and allow monitoring cell viability before in vivo implant, warranting the quality of ESI in situations where longer cultures periods are necessary prior to implantation. After loaded in vitro with stromal cells, microcarriers can be implanted in the subcutaneous tissue of experimental animals as an Ectopic Stroma Implant (ESI) functionally able to rescue lethally irradiated mice. This alternative 3D model allows the functional study of stromal cells. To test stromal cell alterations in our ESI model, we used Cx43 deficient stromal cells. Cx43 is the most abundant Cx in mammalian tissues and one element of gap junction intercellular communication (GJIC) (40). In humans, Cx43 deficiency causes oculo-dental-dysplasia (39,43), a disease characterized by bone malformation. In hematopoietic tissue, Cx43 had been shown to be hyperexpressed in the hematopoietic niche after demand (27). Moreover Cx43 deficient mice have CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 6

Copyright © 2012 Cognizant Communication Corporation reduced number of progenitors in their fetal livers (8). Results with bone marrow chimeras using Cx43+/- host mice suggested that Cx 43 expression in the radioresistant element was important for thymocyte development (37). Also, with the use of IFN induced gene deletion it was shown that after challenge with 5-FU, hematopoietic recovery was impaired and the number of hematopoietic progenitors diminished in experimental mice (41). However, deletion obtained with this model is not stroma specific and affects all BM cells. One possibility to unequivocally confirm the role of Cx43 in the stroma component of the hematopoietic niche would be the use Cx43 deficient cells in our ESI model. Cx43 deficient stroma was generated using short hairpin based specific RNA interference (shRNA) and tested for their hematopoietic support in our ESI model. We show that Cx43 expression in the stroma population is indeed critical to maintain hematopoiesis in vivo, confirming the role of this molecule in the hematopoietic stroma and ascertaining ESI as a suitable model to evaluate individual stromal components.


Copyright © 2012 Cognizant Communication Corporation Materials and Methods

Cell Lines Cell lines were obtained from the Rio de Janeiro Cell Bank (BCRJ, Federal University of Rio de Janeiro, Brazil). Murine S17 BM stroma cells were used according to authorization by Dr K Dorshkind (10). All the experiments were carried out using S17 cells below the 29th passage of the original stock. Viability, assessed by trypan blue exclusion, was always superior to 95% at the beginning of each experiment.

Human Umbilical cord blood and Bone Marrow samples Human umbilical cord blood units and bone marrow samples were obtained after approval of the UCBB of the National Cancer Institute (INCA-Brazil – project number CEP INCA 31/03) and under donor informed consent. UCB was collected according to institutional standard operating procedures. Normal primary BM were obtained and processed as previously described (38).

Flow cytometry analysis CD45+/CD34+ cell enumeration was done with a dual platform protocol and gating strategy similar to the International Society for Cellular Therapy (formerly ISHAGEISCT) guidelines following the method described by Keeney et al (22). KSL cell enumeration was done as described elsewhere (20). Briefly, 2X106 cells/mL were incubated with after staining with anti-c-KIT-APC (2B8 clone, Pharmingen, San Diego,

USA),

anti-SCA-1-PE

(D7

clone)

,

anti-CD3-biotin

(145-2C1

clone,

Pharmingen), anti-CD45/B220-biotin (RA36B2 clone, Phrmingen), TER119-biotin CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 8

Copyright © 2012 Cognizant Communication Corporation (Ly76 clone, Pharmingen), anti-pan-NK-biotin (CD56+CD16, DX clone, Pharmingen), anti-CD11b-biotin (M1/70 clone, Pharmingen) and anti-GR1-PECy5 (LY6C clone, Pharmingen), followed by streptavidin PECy5 (Pharmingen). GJ-mediated intercellular communications between the stroma and hematopoietic cells were monitored by flow cytometry, following the protocol described by Czyz et al. (11) with slight modifications as previously described (38). Briefly, stroma cells were grown to semiconfluence (5 104 cells/cm2) in 24-well plates. They were washed with 0.9% NaCl solution followed by RPMI-1640 culture medium. Stroma monolayers were loaded with 1 M calcein AM (Molecular Probes/ Invitrogen, Eugene, USA) for 2 h, and extensively washed with a serum-free followed by serum-supplemented medium. Leukemic cells were then added in 1 : 1 ratio in relation to stromal cells. After 72 h, cells were trypsinized washed with PBS supplemented with 5% FBS, fixed in 4% paraformaldehyde in PBS and analyzed by flow cytometry. Where indicated, CBX (Sigma, St Louis, MO, USA) was used in the coculture system to inhibit GJmediated cell communication.

Cytopore Microcarriers Cytopore macroporous microcarriers (GE healthcare, São Paulo, Brazil) were hydrated with PBS solution (100mL/g of dry weight) in previously silliconated flasks and autoclaved at 121oC for 20min. These microcarriers were washed with PBS and ressuspended in complete medium (either RPMI or DMEM). Cells were added to the microcarriers at a concentration of 3x104 cells/cm2 of microcarrier area, in 2.0mL cryotubes. These suspensions rested for 2hs at 37oC with 5% CO2. After this time the CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 9

Copyright © 2012 Cognizant Communication Corporation tubes were put under mild agitation (20-30rpm) for 45 min followed by a 15 min period of resting. This 1 hour cycle was repeated over the next 12 hours.

Animals BALB/c mice were bred at the Instituto Nacional de Câncer animal facility (Rio de Janeiro, Brazil). Animals used as hosts in the transplantation protocols were females aged 12 to14 weeks old. All other animals were 8 to 10 weeks. Mice were housed in sterilized micro-isolator cages and were handled according to our institutional guidelines approval. Each transplant group had at least 5 mice.

Generation of connexin 43 silenced S17 cell line Oligonucleotides corresponding to sense and antisense sequence of Cx43 or scrambled shRNA (table S1) of the shRNA+loop were synthesized (Invitrogen, São Paulo, Brazil), annealed, phosphorilated and cloned into XhoI and HPAI and alkaline phosphatase treated pLentiLox pLL3.7 vector (kindly provided by Dr. Luk Van Parijs). Clones were inserted in E. coli XL1, and after confirmation by DNA sequencing, they were transfected into 293T cells using a 3rd generation packing system and VSV tropism. Supernatant containing viral vectors were concentrated and S17 cells were transduced by 48 h exposure to viral stock. By this strategy, three cell lines additional to the original S17 were generated: S17.pLL, transfected with empty pLL vector; S17.SCR, transfected with pLL vector containing Cx43 scrambled sequence, and S17.Cx43, transfected with pLL vector containing the specific sequence for Cx43 inhibition.


Copyright © 2012 Cognizant Communication Corporation

Cobblestone Area Forming Cell (CAFC) assay Stromas were seeded in 96 well plates at 75% confluence, with standard culture medium. Hematopoietic stem cells (mouse bone marrow or human umbilical cord blood) were plated over the stromas in limiting dilution conditions, with 12 repetitions for each different concentration. Co-cultures were left for 5 weeks, with a weekly 50% change of the medium. After this period, each well was analyzed in the microscope for the formation of cobblestone areas. The wells were then checked as positive or negative for the growth of CAFC colonies. Frequency was calculated with the L-calc software (Stem Cell, Vancouver, Canada). In experiments where carbenoxolone (CBX) was used, the medium replacement was supplemented with CBX in experimental plates.

Ectopic Stroma Implant (ESI) model BALB/c recipients received 6 subcutaneous injections of 50µL of a 50% suspension of cytopore microcarrier previously loaded or not with the different stromas as indicated. For hematopoietic recovery studies, one day before transplantation, BALB/c recipients received total body irradiation for myeloablation (850 rads, TH780C irradiator with a cobalt Co 60 [60Co] source). On the next day, recipients received 6 injections of 50µL of a 50% suspension of cytopore microcarriers previously loaded with stroma and 5x106 BALB/C bone marrow cells. Groups consisted of 5-8 mice.

ESI Histological Preparation CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 11

Copyright © 2012 Cognizant Communication Corporation Whenever histological analysis were necessary, ESI and eventually peri-implant tissue were carefully excised from the subcutaneous tissue of euthanized mice with the help of sterile scissors. ESIs were placed inside tissue processing cassettes and fixed in formalin (Formaldehyde 4%). They were routinely embedded in paraffin and processed for Hematoxilin and eosin (H&E) staining

ELISA for Cx43 Immunoenzimatic 96 well plates (Corning, Glendale, USA) were prepared with 100µL of

100ug/mL

rabbit

polyclonal

antibody

against

Cx43,

diluted

in

carbonate/bicarbonate buffer (Na2CO3 0.06M mixed with NaHCO3 0.06M in 1:4 proportion), incubated at 37o C for 2 hours then at 4o C overnight. Plate wells were washed with PBS/Tween 20 0.05% non-fat dry milk 1% for three times. Cell extracts made by treatment of S17 stromas with Tris.Cl 100mmol/L and Triton X-100 0.01% then adjusted to a protein concentration of 100mg/mL. Cells extracts were incubated for 2 hours at room temperature and washed with PBS/Tween/milk. Next, plate wells were incubated with 50µg/mL monoclonal antibody anti-Cx43 (Invitrogen) for 2 hours at room temperature then washed. Detection was made with a TMB chromogenic system (BD Biosciences, Franklin Lakes, USA).

Reading was at 620-

650nm.

Immunocytochemistry Cx43 Cells for immunocytochemistry analysis were cultured in multiplate slides (NUNC, Rochester, USA) until 75% confluence, when cells were fixed in methanol 100% v/v. CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 12

Copyright © 2012 Cognizant Communication Corporation After 10min methanol was drained, slides were dried at room temperature, and washed for 2 hours with PBS/Tween/Milk. Next, slides were incubated with 50µg/mL monoclonal antibody anti-Cx43 (Invitrogen) for 2 hours at room temperature and washed with PBS/Tween/Milk, followed by anti mouse IgG-peroxidase. Detection was made with DAB chromogenic system (BD Biosciences, CA, USA). Statistical Analysis For Cobblestone Area Forming Cell (CAFC) assay statistics were calculated using Lcalc software (Pearson`s statistic). For Ectopic stroma implant (ESI) model and ELISA for Cx43 ANOVA test was used under Prisma Software (Graphpad Prism V5.0, Graphpad Software, La Jolla, USA), with Dunn post-test. Results:

Macroporous Microcarriers are substrates for S17 stromal seeding Macroporous Microcarriers (Cytopore) used in this report are cotton cellulose cross linked matrix covered with Hydrophilic DEAE exchanger for positive charge (18) . They are designed as substrates for high-density culture which allow expansion of available growth surface area for plastic adherent cell lines. Here we used S17 stromal cell line seeded on macroporous microcarriers under agitation for 24 hours. After that the cells can easily be seen under an optical microscope (Figure 1A). The use of vital dyes (Figure 1B) or crystal violet (Figure 1C) allows morphological identification and evaluation of cell viability. In vitro the microcarriers resembles an

ex-vivo explant confirming the viability of the seeded cells. When taken out of the CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 13

Copyright © 2012 Cognizant Communication Corporation suspension culture and set in static culture conditions, cells will migrate, colonize the surrounding area and further give rise to a new monolayer culture (Figure 1D). This finding supports the idea that microcarrier suspension culture keeps cell adhesion properties and viability. Under histological examination (Figure 1E) cells can be seen all over the microcarrier, with mitotic (Figure 1F) and apoptotic (Figure 1G) events suggesting that the dynamic maintenance of a three-dimensional culture is reproducible in this model of cell culture.

Ectopic stroma implant model (ESI): Macroporous Microcarriers loaded with S17 are kept in vivo and support local hematopoiesis After being sure that the microcarriers chosen could provide an appropriate environmental support for stromal cell growth, we injected microcarriers containing the S17 cell line into the subcutaneous tissue of a BALB/c mouse syngeneic to the stroma. Figure 2 illustrates the external appearance of such

transplants and

compares transplants of microcarriers where S17 cells are present or not (figure 2A and B). When S17 stromal cells are present, a transplant nodule can be seen and measured (figure 2B). However, in the absence of S17 cells, the microcarriers can hardly be seen and in 2 weeks they become barely detectable (figure 2A and 2F). After 4 to 6 weeks, empty microcarriers are absorbed not being detectable at all. Macro anatomically, S17 full microcarriers seem to be surrounded by a membrane resembling a vascularized granuloma (figure 2B). At histological level, it is surrounded by mononuclear infiltrate and blood vessels can be observed on the edge of the microcarrier area (figure 2C). The S17 positive microcarriers are fully CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 14

Copyright © 2012 Cognizant Communication Corporation populated and the presence of hematopoietic cells, especially granulocytes, in different maturation stages can be observed (Figure 2D and E). These results suggest that transplanted microcarriers alone are not enough to support hematopoiesis in vivo and that in the absence of stromal cells they are absorbed without any evident damage to the host. The stromal cells transplanted inside the microcarriers might be essential to attract and support hematopoietic progenitor development. These progenitors can differentiate inside the stroma loaded microcarriers allowing the in vivo study of the hematopoietic supporting activity of the stroma, mimicking an ectopic bone marrow.

Short Hairpin inhibition of Cx43 expression in S17 stromal cell lines inhibit hematopoietic support in vitro We chose Connexin 43 (Cx43) as target molecule to test the viability of our ESI model in studies of single stromal elements important for hematopoietic support. Cx43 is the most abundant Cx in mammalian tissues and is highly expressed in the hematopoietic stem cell niche (27). Moreover, Cx43 has been suggested to be critically important to maintain hematopoietic homeostasis in different reports (8,37). Finally, it has been recently shown that Cx43 is an important regulator of CXCL12 secretion indicating an important role for this molecule in hematopoietic stem cell niche maintenance (47). To address the specific role of Cx43 in stromal cells, we used Cx43 shRNA silenced stromas. shRNA was transduced to the stromal cell line S17 using the lentiviral vector CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 15

Copyright © 2012 Cognizant Communication Corporation pLL 3.7 lentilox carrying previously described shRNA sequences targeting Cx43 (48). Silencing with shRNA for Cx43 inhibited protein expression evaluated by Cx43 specific ELISA and immunocytochemistry (supplementary figure 1A and B), without affecting doubling time or phosphatase activity (supplementary figure 1C and D). This indicates that at least no major modifications on S17 cell line behavior were taking place after lentiviral transduction. To study the effect of Cx43 in GJIC, immature hematopoietic progenitors (cKit+sca1+lin- or KSL) were co-cultured with calcein loaded stromal cells and the number of calcein+ KSL after the co-culture period was addressed. GJIC was compromised between stroma and hematopoietic progenitor cells as seen by a reduction from 80% to 30% in KSL communicating cell (Calcein+) co-cultured with Cx43 silenced cultures compared to control (supplementary figure 1E). These results suggest that Cx43 is an important mediator of cell-cell communication between bone marrow stroma and hematopoietic progenitors. The S17 stromal cell line silenced for Cx43 (S17.Cx43) was tested for its functionality regarding hematopoietic support. Stromas were cultured for 5 weeks with bone marrow cells in limiting dilution conditions. CAFC colonies from S17.Cx43 were less than one fifth that of control stromas, indicating that Cx43 is indeed important to maintain immature hematopoietic progenitors (Figure 3A). This result confirms previous results from Cancelas’ group using fetal liver stroma cell lines from Cx43 KO mice (8). We had previously shown that GJIC was important to keep leukemic cells in a quiescent state. We postulated that this would be important to maintain stem cells CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 16

Copyright © 2012 Cognizant Communication Corporation and in the absence of Cxs, differentiation would take place (38) . This correlates with the results shown in figure 3B where smaller numbers of immature KSL+ hematopoietic progenitors are recovered from S17.Cx43 supporting cultures. In fact, the total amount of mature cells generated when the supporting stroma is deficient in Cx43 expression is higher when compared to control cultures (figure 3C left panel). Moreover, with time the number of cells generated on a weekly basis drops more acutely in the absence of Cx43 than in the presence (figure 3C right panel). By the 5th week basically no nucleated cells come out of Cx43 silenced cultures and the stroma is devoid of CAFCs (supplementary figure 2) Altogether, these results indicate that the presence of Cx43 in the hematopoietic supporting stroma is indeed critical to maintain hematopoietic stem cell progenitors, at least in vitro.

ESI can be used to reconstitute hematopoiesis and to address the role of single stromal element in hematopoiesis in vivo ESI empty microcarriers or carriers loaded with the stromal cells S17, S17.pLL, S17.Scr or S17.Cx43 were implanted in the subcutaneous tissue of regular or irradiated mice to evaluate hematopoietic supporting activities. ESI implants were performed at multiple sites in each individual mouse. At the site of injection, a nodule is formed as shown above (figure 2B). Basically, one hundred percent of injected sites develop into nodules after 2 weeks, as long as the microcarriers are loaded with functional S17 stromas (Figure 2F). However, the CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 17

Copyright © 2012 Cognizant Communication Corporation nodule size, measured at two weeks after injection, is dependent on the presence of functional stroma and empty carriers as well as ESI carrying Cx43 deficient stromas are smaller than ESI carrying Cx43 sufficient cells (Figure 4A), suggesting impairment on the hematopoietic support activity in the absence of Cx43. One advantage of ESI is the possibility of recovery which enables the analysis of the ESI inner content. ESIs were recovered and analyzed by histopathology. In the presence of control stromas, an intense hematopoietic activity is observed (Figure 4B) which, at a macroscopic level is seen as larger subcutaneous nodules (Figure 4A). However, in the presence of S17.Cx43 the ESI is devoid of hematopoietic cells confirming the inability to either sustain hematopoiesis or attract progenitor cells (Figure 4B). To better investigate both, the hematopoietic reconstitution potential of the ESI and Cx43 dependence of the stroma, BALB/c mice were irradiated and reconstituted with microcarriers loaded with stroma and BALB/c total bone marrow cells. By the end of a 6 week follow up period groups containing S17 control stroma achieved an overall survival of nearly 70% against the 40% observed for the group receiving either empty microcarriers or microcarriers loaded with S17.Cx43 stroma (Figure 4C). Hematological reconstitution was assessed through total nucleated blood cell count. After 2 weeks, all groups showed aplasia, even though S17.Cx43 group had a significant higher blood count compared to other groups (3% of control BALB/c leukogram against 1% in the control Cx43 positive). By the 4th week, no significant difference was seen among all groups although Cx43 silenced group tend to be CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 18

Copyright © 2012 Cognizant Communication Corporation smaller By the 6th week, lack of Cx43 lead to lower blood counts in reference to all other groups (Figure 4D). This pattern is similar to that seen in vitro, where an initial proliferation boom followed by the extinction of cell production was observed when the stroma is deficient in Cx43 (figure 3C) suggesting a role in stem cell maintenance for Cx43.


Copyright © 2012 Cognizant Communication Corporation Discussion Given the complexity of the stem cell niche (34,51,60) we wish to design a simple strategy to enable the study of stromal elements in vivo. Our model was based on the in vivo use of cellulose matrix macroporous microcarriers (Cytopore) developed to increase tissue culture area in vitro. Empty microcarriers implanted in the subcutaneous tissue are rapidly reabsorbed suggesting that it can be employed without major risks/toxicity to the host. This could represent an advantage over Ceramic HA/TCP supports. The latter has been widely used in orthopedics and experimental hematology where it serves as support to bone (26,33) and bone marrow formation (36,58). HA particles are bigger than the Cytopore used here and its absorption is very slow, usually accompanied by bone formation or replacement (2). Another advantage of using cellulose microcarriers is the possibility to monitor cell viability in vitro prior to the in vivo studies, warranting the quality of the biological material to be implanted. Stromal cells can easily be identified inside the microcarriers where they are able to proliferate, apoptose, and support hematopoiesis (data not shown). When implanted in the subcutaneous tissue, cellulose microcarriers provide different outcomes determined by the feeder stroma. If the microcarriers are used empty, without S17 cells, they are rapidly absorbed and within 4 weeks no implant is left. These results indicate that microcarriers by themselves are afunctional and atoxic when used in vivo. It also tells us that the stroma, in this case, prevents a fast


Copyright © 2012 Cognizant Communication Corporation absorbance. This could be related to the fact that mesenquimal cells are potent immunossupressors (42) and in fact S17 cell line has mesenquimal characteristics. The histological examination of an ESI shows organized tissue surrounded by inflammatory mononuclear cells limiting the implant externally, resembling a granuloma. Some blood vessels are observed in the outer layer. It is important to note that the micro-structure of the microcarriers resembles a BM trabeculi with hematopoietic precursors in different developmental stages being observed, especially neutrophils, indicating that progenitor cells migrate to and differentiate inside the implant. Besides, when tested in reconstitution experiments ESI were able to rescue more than 70% of animals from death, at least 30% more than control (empty microcarriers). These observations encouraged us to test if this model was suitable to in vivo study of stromal cells modifications. The molecule chosen was Cx43, as its importance in hematopoiesis had been pointed by several groups (8,37,41), but its role on the stroma had not been unequivocally addressed in vivo Cx43 deficient stroma was generated using shRNA and tested for their hematopoietic support. In vitro, Cx43 deficient cells do not couple to KSL and have diminished CAFC support ability. Moreover , in Dexter’s type cultures (13) early differentiation ascertained by increased numbers of floating cells is accompanied by decreased generative capacity, or exhaustion.



In vivo, Cx43 decificient stromas were not colonized by hematopoietic cells. This could be due to Cx43 dependent migration of hematopoietic progenitors. However, when ESI containing Cx43 deficient stroma and bone marrow cells were performed, impairment in maintenance or differentiation was evident by histopathology. Moreover, Cx43 deficient ESI were not as efficient as Cx43 sufficient ESI to rescue irradiated mice. When total nucleated cell were examined in the peripheral blood, a more efficient reconstitution is seen early (3% against 1% of non- irradiated controls) .This early recovery is hampered by important loss on blood counts. At 4 weeks after implant there is no significant difference among the various groups. However, by the 6th week mice reconstituted with Cx43 deficient ESI are severely cytopenic indicating the importance of this molecule to maintain the stem cells. Various mechanisms had been proposed to explain the role of Cx43 in cell fate (8,12,17,19,30,56) and this had gained attention with identification of mutations related to human oculo-dental-dysplasia (39). More than 60 Gja1 mutations had been reported with several skeletal malformations (28,39). In experimental models hematological defects appear as the animals age raising the possibility of immunologic defects either in senior years of after demand (15,28). Very recently Cx43 and Cx45 were shown to be important for CXCL12 secretion (47), a chemokine critical for osteoblastic/hematopoietic niche organization (53). In the absence of CXCL12, an increase in numbers of short term HSCs and decreased long-term HSCs was observed (53). The Cx43 regulation of CXCL12 fits well with our results showing that in Dexter`s type cultures, differentiation and exhaustion are faster in the CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 22

Copyright © 2012 Cognizant Communication Corporation absence

of

Cx43.

Moreover,

faster

reconstitutions

with

abrupt

decay

of

hematopoiesis support is seen in vivo using ESI, showing the importance of Cx43 in the stroma and validating ESI as a model system to study the stromal elements of the hematopoietic niche In summary, the proposed ESI used here represents a simple alternative way to reconstitute hematopoiesis in vivo using a 3D stroma implant model, specially in the case of stroma related diseases. As important as an ectopic hematopoietic device, it is a support for stromal cells that can receive hematopoietic cell and promote hematopoiesis. As so, it provides a mean to study and manipulate individual components of hematopoietic supportive cells present in the bone marrow environment in vivo.


Copyright © 2012 Cognizant Communication Corporation Acknowledgments We thank Romulo Areal Braga for helping with figures preparation, Antonio Carlos Campos de Carvalho and Regina Goldenberg for providing the anti-Cx43 antibodies and Ana Carolina Leal and Leonardo Chicaybam for assistance with shRNA techniques. This work was supported by CNPq, FAPERJ, INCT Câncer, Ministry of Health/INCA and Swiss Bridge Foundations grants. Authorship contributions: Flávio Henrique Paraguassú-Braga – designed and performed the experiments, analyzed the data and wrote the paper ;Ana Paula Gregório Alves

-performed

experiments; Isabela Maria Alvim Andrade Santos – performed experiments; Martin Bonamino – designed shRNA experiments, analyzed data; Adriana Bonomo – designed experiments, analyzed data and wrote the paper Conflict of interest: The authors declare no conflict of interest Links to supplemental matherial: Table S1: http://issuu.com/mbona/docs/supplement-paraguassu-braga-et-al1?mode=a_p Supplementary Figure 1: http://issuu.com/mbona/docs/supplement-paraguassu-braga-etal1?mode=a_p Supplementary Figures 2: http://issuu.com/mbona/docs/supplement-paraguassu-braga-etal1?mode=a_p




Copyright © 2012 Cognizant Communication Corporation References:

1.

2. 3. 4. 5. 6.

7.

8.

9.

10. 11. 12.

Aizawa, S.; Yaguchi, M.; Nakano, M.; Toyama, K.; Inokuchi, S.; Imai, T.; Yasuda, M.; Nabeshima, R.; Handa, H. Hematopoietic supportive function of human bone marrow stromal cell lines established by a recombinant SV40adenovirus vector. Exp. Hematol. 22(6):482-487; 1994. Baas, J. Adjuvant therapies of bone graft around non-cemented experimental orthopedic implants stereological methods and experiments in dogs. Acta Orthop. Suppl. 79(330):1-43; 2008. Bacigalupo, A.; Palandri, F. Management of acute graft versus host disease (GvHD). Hematol. J. 5(3):189-196; 2004. Bagley, J.; Rosenzweig, M.; Marks, D. F.; Pykett, M. J. Extended culture of multipotent hematopoietic progenitors without cytokine augmentation in a novel three-dimensional device. Exp. Hematol. 27(3):496-504; 1999. Banu, N.; Rosenzweig, M.; Kim, H.; Bagley, J.; Pykett, M. Cytokine-augmented culture of haematopoietic progenitor cells in a novel three-dimensional cell growth matrix. Cytokine 13(6):349-358; 2001. Barton-Burke, M.; Dwinell, D. M.; Kafkas, L.; Lavalley, C.; Sands, H.; Proctor, C.; Johnson, E. Graft-versus-host disease: a complex long-term side effect of hematopoietic stem cell transplant. Oncology (Williston Park) 22(11 Suppl. Nurse Ed.):31-45; 2008. Borojevic, R.; Roela, R. A.; Rodarte, R. S.; Thiago, L. S.; Pasini, F. S.; Conti, F. M.; Rossi, M. I.; Reis, L. F.; Lopes, L. F.; Brentani, M. M. Bone marrow stroma in childhood myelodysplastic syndrome: composition, ability to sustain hematopoiesis in vitro, and altered gene expression. Leuk. Res. 28(8):831844; 2004. Cancelas, J. A.; Koevoet, W. L.; de Koning, A. E.; Mayen, A. E.; Rombouts, E. J.; Ploemacher, R. E. Connexin-43 gap junctions are involved in multiconnexin-expressing stromal support of hemopoietic progenitors and stem cells. Blood 96(2):498-505; 2000. Cavazzana-Calvo, M.; Andre-Schmutz, I.; Dal Cortivo, L.; Neven, B.; HaceinBey-Abina, S.; Fischer, A. Immune reconstitution after haematopoietic stem cell transplantation: obstacles and anticipated progress. Curr. Opin. Immunol. 21(5):544-548; 2009. Collins, L. S.; Dorshkind, K. A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis. J. Immunol. 138(4):1082-1087; 1987. Czyz, J.; Irmer, U.; Schulz, G.; Mindermann, A.; Hulser, D. F. Gap-junctional coupling measured by flow cytometry. Exp. Cell Res. 255(1):40-46; 2000. DeSano, J. T.; Xu, L. MicroRNA regulation of cancer stem cells and therapeutic implications. AAPS J. 11(4):682-692; 2009. CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 26

Copyright © 2012 Cognizant Communication Corporation 13. 14. 15.

16.

17.

18.

19. 20.

21.

22.

23.

Dexter, T. M. Haemopoiesis in long-term bone marrow cultures. A review. Acta Haematol. 62(5-6):299-305; 1979. Drobyski, W. R. Evolving strategies to address adverse transplant outcomes associated with T cell depletion. J. Hematother. Stem Cell Res. 9(3):327-337; 2000. Flenniken, A. M.; Osborne, L. R.; Anderson, N.; Ciliberti, N.; Fleming, C.; Gittens, J. E.; Gong, X. Q.; Kelsey, L. B.; Lounsbury, C.; Moreno, L.; Nieman, B. J.; Peterson, K.; Qu, D.; Roscoe, W.; Shao, Q.; Tong, D.; Veitch, G. I.; Voronina, I.; Vukobradovic, I.; Wood, G. A.; Zhu, Y.; Zirngibl, R. A.; Aubin, J. E.; Bai, D.; Bruneau, B. G.; Grynpas, M.; Henderson, J. E.; Henkelman, R. M.; McKerlie, C.; Sled, J. G.; Stanford, W. L.; Laird, D. W.; Kidder, G. M.; Adamson, S. L.; Rossant, J. A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia. Development 132(19):4375-4386; 2005. Fouillard, L.; Bensidhoum, M.; Bories, D.; Bonte, H.; Lopez, M.; Moseley, A. M.; Smith, A.; Lesage, S.; Beaujean, F.; Thierry, D.; Gourmelon, P.; Najman, A.; Gorin, N. C. Engraftment of allogeneic mesenchymal stem cells in the bone marrow of a patient with severe idiopathic aplastic anemia improves stroma. Leukemia 17(2):474-476; 2003. Fruscione, F.; Scarfi, S.; Ferraris, C.; Bruzzone, S.; Benvenuto, F.; Guida, L.; Uccelli, A.; Salis, A.; Usai, C.; Jacchetti, E.; Ilengo, C.; Scaglione, S.; Quarto, R.; Zocchi, E.; De Flora, A. Regulation of Human Mesenchymal Stem Cell Functions by an Autocrine Loop Involving NAD(+) Release and P2Y11Mediated Signaling. Stem Cells Dev. 20(7):1183-1198; 2011. GE. Cytopore™ 1 and Cytopore™ 2. 2011 [cited 2011 08/10/2011]; Available from: http://www.gelifesciences.com/aptrix/upp01077.nsf/content/Products?OpenD ocument&parentid=666921&moduleid=167173 Guo, S.; Scadden, D. T. A microRNA regulating adult hematopoietic stem cells. Cell Cycle 9(18):3637-3638. Hirabayashi, Y.; Yoon, B. I.; Tsuboi, I.; Huo, Y.; Kodama, Y.; Kanno, J.; Ott, T.; Trosko, J. E.; Inoue, T. Membrane channel connexin 32 maintains Lin(-)/ckit(+) hematopoietic progenitor cell compartment: analysis of the cell cycle. J. Membr. Biol. 217(1-3):105-113; 2007. Kagan, W. A.; Ascensao, J. A.; Pahwa, R. N.; Hansen, J. A.; Goldstein, G.; Valera, E. B.; Incefy, G. S.; Moore, M. A.; Good, R. A. Aplastic anemia: presence in human bone marrow of cells that suppress myelopoiesis. Proc. Natl. Acad. Sci. U S A 73(8):2890-2894; 1976. Keeney, M.; Chin-Yee, I.; Weir, K.; Popma, J.; Nayar, R.; Sutherland, D. R. Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering. Cytometry 34(2):61-70; 1998. Kim, H. S.; Lim, J. B.; Min, Y. H.; Lee, S. T.; Lyu, C. J.; Kim, E. S.; Kim, H. O. Ex vivo expansion of human umbilical cord blood CD34+ cells in a collagen bead-containing 3-dimensional culture system. Int. J. Hematol. 78(2):126132; 2003. CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 27

Copyright © 2012 Cognizant Communication Corporation 24. 25. 26.

27. 28. 29.

30.

31. 32. 33. 34.

35.

36.

Kirouac, D. C.; Zandstra, P. W. Understanding cellular networks to improve hematopoietic stem cell expansion cultures. Curr. Opin. Biotechnol. 17(5):538547; 2006. Koestenbauer, S.; Zisch, A.; Dohr, G.; Zech, N. H. Protocols for hematopoietic stem cell expansion from umbilical cord blood. Cell Transplant. 18(10):10591068; 2009. Krebsbach, P. H.; Kuznetsov, S. A.; Satomura, K.; Emmons, R. V.; Rowe, D. W.; Robey, P. G. Bone formation in vivo: comparison of osteogenesis by transplanted mouse and human marrow stromal fibroblasts. Transplantation 63(8):1059-1069; 1997. Krenacs, T.; Rosendaal, M. Connexin43 gap junctions in normal, regenerating, and cultured mouse bone marrow and in human leukemias: their possible involvement in blood formation. Am. J. Pathol. 152(4):993-1004; 1998. Laird, D. W. Closing the gap on autosomal dominant connexin-26 and connexin-43 mutants linked to human disease. J. Biol. Chem. 283(6):29973001; 2008. Lange, C.; Brunswig-Spickenheier, B.; Cappallo-Obermann, H.; Eggert, K.; Gehling, U. M.; Rudolph, C.; Schlegelberger, B.; Cornils, K.; Zustin, J.; Spiess, A. N.; Zander, A. R. Radiation rescue: mesenchymal stromal cells protect from lethal irradiation. PLoS One 6(1):e14486; 2011. Lee, K. M.; Kwon, J. Y.; Lee, K. W.; Lee, H. J. Ascorbic acid 6-palmitate suppresses gap-junctional intercellular communication through phosphorylation of connexin 43 via activation of the MEK-ERK pathway. Mutat. Res. 660(1-2):51-56; 2009. Liu, S. P.; Fu, R. H.; Yu, H. H.; Li, K. W.; Tsai, C. H.; Shyu, W. C.; Lin, S. Z. MicroRNAs regulation modulated self-renewal and lineage differentiation of stem cells. Cell Transplant. 18(9):1039-1045; 2009. Madlambayan, G. J.; Rogers, I.; Casper, R. F.; Zandstra, P. W. Controlling culture dynamics for the expansion of hematopoietic stem cells. J. Hematother. Stem Cell Res. 10(4):481-492; 2001. Mankani, M. H.; Kuznetsov, S. A.; Fowler, B.; Kingman, A.; Robey, P. G. In vivo bone formation by human bone marrow stromal cells: effect of carrier particle size and shape. Biotechnol. Bioeng. 72(1):96-107; 2001. Mendez-Ferrer, S.; Michurina, T. V.; Ferraro, F.; Mazloom, A. R.; Macarthur, B. D.; Lira, S. A.; Scadden, D. T.; Ma'ayan, A.; Enikolopov, G. N.; Frenette, P. S. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466(7308):829-834. Meuleman, N.; Tondreau, T.; Ahmad, I.; Kwan, J.; Crokaert, F.; Delforge, A.; Dorval, C.; Martiat, P.; Lewalle, P.; Lagneaux, L.; Bron, D. Infusion of mesenchymal stromal cells can aid hematopoietic recovery following allogeneic hematopoietic stem cell myeloablative transplant: a pilot study. Stem Cells Dev. 18(9):1247-1252; 2009. Miura, Y.; Gao, Z.; Miura, M.; Seo, B. M.; Sonoyama, W.; Chen, W.; Gronthos, S.; Zhang, L.; Shi, S. Mesenchymal stem cell-organized bone marrow CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 28


37. 38.

39.

40. 41.

42. 43.

44.

45.

46. 47.

48.

elements: an alternative hematopoietic progenitor resource. Stem Cells 24(11):2428-2436; 2006. Montecino-Rodriguez, E.; Leathers, H.; Dorshkind, K. Expression of connexin 43 (Cx43) is critical for normal hematopoiesis. Blood 96(3):917-924; 2000. Paraguassu-Braga, F. H.; Borojevic, R.; Bouzas, L. F.; Barcinski, M. A.; Bonomo, A. Bone marrow stroma inhibits proliferation and apoptosis in leukemic cells through gap junction-mediated cell communication. Cell Death Differ. 10(9):1101-1108; 2003. Paznekas, W. A.; Karczeski, B.; Vermeer, S.; Lowry, R. B.; Delatycki, M.; Laurence, F.; Koivisto, P. A.; Van Maldergem, L.; Boyadjiev, S. A.; Bodurtha, J. N.; Jabs, E. W. GJA1 mutations, variants, and connexin 43 dysfunction as it relates to the oculodentodigital dysplasia phenotype. Hum. Mutat. 30(5):724733; 2009. Peracchia, C., ed. Gap Junctions: Molecular Basis of Cell Communication in Health and Disease. San Diego: Academic Press; 2000. Presley, C. A.; Lee, A. W.; Kastl, B.; Igbinosa, I.; Yamada, Y.; Fishman, G. I.; Gutstein, D. E.; Cancelas, J. A. Bone marrow connexin-43 expression is critical for hematopoietic regeneration after chemotherapy. Cell Commun. Adhes. 12(5-6):307-317; 2005. Rasmusson, I. Immune modulation by mesenchymal stem cells. Exp. Cell Res. 312(12):2169-2179; 2006. Roscoe, W.; Veitch, G. I.; Gong, X. Q.; Pellegrino, E.; Bai, D.; McLachlan, E.; Shao, Q.; Kidder, G. M.; Laird, D. W. Oculodentodigital dysplasia-causing connexin43 mutants are non-functional and exhibit dominant effects on wildtype connexin43. J. Biol. Chem. 280(12):11458-11466; 2005. Rosenzweig, M.; Pykett, M.; Marks, D. F.; Johnson, R. P. Enhanced maintenance and retroviral transduction of primitive hematopoietic progenitor cells using a novel three-dimensional culture system. Gene Ther. 4(9):928936; 1997. Rossi, M. I.; Barros, A. P.; Baptista, L. S.; Garzoni, L. R.; Meirelles, M. N.; Takiya, C. M.; Pascarelli, B. M.; Dutra, H. S.; Borojevic, R. Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration. Braz. J. Med. Biol. Res. 38(10):1455-1462; 2005. Sauvageau, G.; Iscove, N. N.; Humphries, R. K. In vitro and in vivo expansion of hematopoietic stem cells. Oncogene 23(43):7223-7232; 2004. Schajnovitz, A.; Itkin, T.; D'Uva, G.; Kalinkovich, A.; Golan, K.; Ludin, A.; Cohen, D.; Shulman, Z.; Avigdor, A.; Nagler, A.; Kollet, O.; Seger, R.; Lapidot, T. CXCL12 secretion by bone marrow stromal cells is dependent on cell contact and mediated by connexin-43 and connexin-45 gap junctions. Nat. Immunol. 12(5):391-398. Shao, Q.; Wang, H.; McLachlan, E.; Veitch, G. I.; Laird, D. W. Downregulation of Cx43 by retroviral delivery of small interfering RNA promotes an aggressive breast cancer cell phenotype. Cancer Res. 65(7):2705-2711; 2005. CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 29

Copyright © 2012 Cognizant Communication Corporation 49.

50. 51. 52. 53.

54.

55. 56. 57. 58.

59. 60.

Sharma, S.; Gurudutta, G. U.; Satija, N. K.; Pati, S.; Afrin, F.; Gupta, P.; Verma, Y. K.; Singh, V. K.; Tripathi, R. P. Stem cell c-KIT and HOXB4 genes: critical roles and mechanisms in self-renewal, proliferation, and differentiation. Stem Cells Dev. 15(6):755-778; 2006. Sorrentino, B. P. Clinical strategies for expansion of haematopoietic stem cells. Nat. Rev. Immunol. 4(11):878-888; 2004. Taichman, R. S. Blood and bone: two tissues whose fates are intertwined to create the hematopoietic stem-cell niche. Blood 105(7):2631-2639; 2005. Tung, S. S.; Parmar, S.; Robinson, S. N.; De Lima, M.; Shpall, E. J. Ex vivo expansion of umbilical cord blood for transplantation. Best Pract. Res. Clin. Haematol. 23(2):245-257; 2010. Tzeng, Y. S.; Li, H.; Kang, Y. L.; Chen, W. C.; Cheng, W. C.; Lai, D. M. Loss of Cxcl12/Sdf-1 in adult mice decreases the quiescent state of hematopoietic stem/progenitor cells and alters the pattern of hematopoietic regeneration after myelosuppression. Blood 117(2):429-439; 2011. Walenda, T.; Bokermann, G.; Ventura Ferreira, M. S.; Piroth, D. M.; Hieronymus, T.; Neuss, S.; Zenke, M.; Ho, A. D.; Muller, A. M.; Wagner, W. Synergistic effects of growth factors and mesenchymal stromal cells for expansion of hematopoietic stem and progenitor cells. Exp. Hematol. 39(6):617-628; 2011. Wang, Y.; Nathanson, L.; McNiece, I. K. Differential Hematopoietic Supportive Potential and Gene Expression of Stroma Cell Lines from Midgestation Mouse Placenta and Adult Bone Marrow. Cell Transplant. 20(5):707-726; 2011 Wang, Y.; Russell, I.; Chen, C. MicroRNA and stem cell regulation. Curr. Opin. Mol. Ther. 11(3):292-298; 2009. Weissman, I. L. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science 287(5457):1442-1446; 2000. Yamaza, T.; Miura, Y.; Akiyama, K.; Bi, Y.; Sonoyama, W.; Gronthos, S.; Chen, W.; Le, A.; Shi, S. Mesenchymal stem cell-mediated ectopic hematopoiesis alleviates aging-related phenotype in immunocompromised mice. Blood 113(11):2595-2604; 2009. Zhang, C. C.; Lodish, H. F. Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. Blood 105(11):4314-4320; 2005. Zhang, J.; Niu, C.; Ye, L.; Huang, H.; He, X.; Tong, W. G.; Ross, J.; Haug, J.; Johnson, T.; Feng, J. Q.; Harris, S.; Wiedemann, L. M.; Mishina, Y.; Li, L. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 425(6960):836-841; 2003.


Copyright © 2012 Cognizant Communication Corporation Figure Legends:

Figure 1:

S17 stromal cells can be visualized inside the microcarriers

under inverted microscopy and by histological examination S17 stroma was seeded in microspheres incubated in cryotube vials in a density of 5x103-1x104 cells/cm2 for 7 days. Medium was changed every other day. (A) Aspect of cytopore microcarrier loaded with S17 cells under standard culture conditions by contrast phase microscopy; (B). Same optical field as in A under fluorescent microscopy, where viable cells were stained with calcein AM vital dye. (C) Cytopore were sampled and fixed with 10% paraformaldehyde and stained with violet crystal (D) Cytopore under continuous agitation cultures were sampled and put in static cultures for 7 days. Viable cells migrate and grow as explants cultures. Leica DMIL inverted microscope, 10x40x/0.50 with sony cybershot DSC-W310. (E) S17 stromal cells were seeded as previously mentioned and cultured on our standard homogenization system for 5 days. After that they were fixed and processed for standard H&E histological routine and submitted to microscopic examination (10x40x/0.65). (F) Zoom in of a section of cytopore microccarrier showing mitosis (arrow) and (G) apoptotic cell (arrow). Nikon Eclipse E20, and 10x100x/1.25 with sony cybershot DSC-W310.

Figure 2 Establishment of stroma implant in a microcarrier support-Ectopic

Stroma Implant Model: (A) development of ESI depends on the presence of stroma: a cytopore microcarrier suspension loaded (right side) or not (left side) with stromal cells was CT-0580 Cell Transplantation Epub; provisional acceptance 11/22/2011 31

Copyright © 2012 Cognizant Communication Corporation injected in the subcutaneous tissue of BALB/c mouse for 6 weeks. Microcarriers implanted free of stroma cells do not develop (B) Macroscopic aspect of subcutaneous tissue injected with stroma containing cytopore, showing vascular development towards the ESI. (C) Panoramic histological view of skin and subcutaneous compartment (H&E). Leica DMIL inverted microscope, 20x/0.30 with sony cybershot DSC-W310. (D) Section of subcutaneous area showing trabeculae-like cytopore microcarrier where fibroblastic cells can be seen. Leica DMIL inverted microscope, 40x/0.50 with sony cybershot DSC-W310. (E) Another section of subcutaneous area demonstrating not only fibroblastic cells, but also blood vessels and granulocytes. Mature (arrow) and immature granulocytes (arrow head) is indicated. Leica DMIL inverted microscope, 10X40x/0.50 with sony cybershot DSC-W310. (F) Microcarriers were injected ate 6 different sites in each mouse. The number of sites where a nodule was formed was counted after 4 weeks. Notice that stroma-free microcarriers hardly develop into nodes

Figure 3 Cx43 silenced stroma cannot sustain the maintenance of immature

hematopoietic progenitors: (A) Diminished hematopoietic progenitor support activity in the absence of Cx43. Total BALB/c mouse bone marrow was co-cultured with the various S17 stromas in limiting dilution conditions to ascertain CAFC as described in material and methods. Analysis was performed using L-calc software (P