A New Approach to Expand Cord Blood Derived Hematopoietic Stem Cells, Using Bioengineered Human Fetal Liver Tissue 3D-Constructs

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3097-3097
Author(s):  
Saloomeh Mokhtari ◽  
Pedro Baptista ◽  
Dipen Vyas ◽  
Charles Jordan Freeman ◽  
Emma Moran ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Stromal Cells ◽  
Culture System ◽  
Fetal Liver ◽  
Rapid Expansion ◽  
Hematopoietic Stem ◽  
3D Cultures

Abstract Despite advances in ex-vivo expansion of cord blood-derived hematopoietic stem/progenitor cells (CB-HSPC), challenges still remain regarding the ability to obtain, from a single unit, sufficient numbers of both long- and short-term repopulating cells, capable of functional engraftment, to enable the treatment of an adolescent or adult patient. We have previously shown that CB-HSPC can be effectively expanded in a 2D serum-free culture system, using a feeder layer of adult human bone marrow-derived stromal cells; still, the percentage of the most primitive stem cells decreased with time. Because, during development, the fetal liver is the main site of HSC expansion and differentiation, we hypothesized that efficient expansion of functional HSPC could be achieved in vitro under more physiologic conditions provided by surrogate fetal liver microenvironments. Therefore, we compared bioengineered liver constructs made from a natural 3D liver extracellular matrix (3DExM) seeded with hepatoblasts (HB), fetal liver-derived stromal cells (FLSC), or bone marrow-derived stromal cells (BMSC), with a 2D culture system using FLSC or BMSC. Overall, 2D culture systems generated a higher yield of mature blood cells by day 14, mostly within the myelomonocytic lineages, with fold increases in total cell number in 2D-HB, 2D-FLSC, and 2D-BMSC cultures of 1145, 16151, and 229, respectively, while 3D cultures generated fold increases of 94.3, 492, and 110, respectively. Nevertheless, the output and expansion of more primitive HSPC was significantly higher in 3D cultures, as determined by flow cytometry and colony-forming assays. Specifically, in 3D-HB cultures, the percentage of CD34+CD38- cells increased by 100% at day 2, while 3D-FLSC and 3D-BMSC cultures each supported a 90% increase in the percentage of CD34+CD38- cells during this period. By contrast, all of the 2D cultures experienced a 30% decrease in the percentage of CD34+CD38- cells during this same time period. In addition, only 3D conditions maintained CD34+CD38- cells until day 12 of culture. Colony-forming assays demonstrated that the CFU-GEMM output was higher in 3D cultures when compared with their 2D counterpart. Among the 3D cultures, the 3D-HB cultures had the highest number of CFU-GEMM at day 2 and day 4. In conclusion, we demonstrate that by integrating biological components in vitro to obtain structures that contain all the necessary elements to mimic the fetal liver microenvironmental niches, which are known to promote rapid expansion of HSC during development, we were able to achieve significant expansion and maintenance of CD34+CD38- cells. In addition, since little is known about fetal liver niches that support HSPC expansion, the 3D constructs will provide, for the first time, a model system in which to dissect the role of the individual cellular and matrix niche components in supporting CB HSPC maintenance, expansion, and differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Molecular Modulation of Fetal Liver Hematopoietic Stem Cell Mobilization into Fetal Bone Marrow in Mice

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Huihong Zeng ◽  
Jiaoqi Cheng ◽  
Ying Fan ◽  
Yingying Luan ◽  
Juan Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Vascular Endothelial Cells ◽  
Fetal Liver ◽  
Bone Marrow Niche ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fetal Bone

Development of hematopoietic stem cells is a complex process, which has been extensively investigated. Hematopoietic stem cells (HSCs) in mouse fetal liver are highly expanded to prepare for mobilization of HSCs into the fetal bone marrow. It is not completely known how the fetal liver niche regulates HSC expansion without loss of self-renewal ability. We reviewed current progress about the effects of fetal liver niche, chemokine, cytokine, and signaling pathways on HSC self-renewal, proliferation, and expansion. We discussed the molecular regulations of fetal HSC expansion in mouse and zebrafish. It is also unknown how HSCs from the fetal liver mobilize, circulate, and reside into the fetal bone marrow niche. We reviewed how extrinsic and intrinsic factors regulate mobilization of fetal liver HSCs into the fetal bone marrow, which provides tools to improve HSC engraftment efficiency during HSC transplantation. Understanding the regulation of fetal liver HSC mobilization into the fetal bone marrow will help us to design proper clinical therapeutic protocol for disease treatment like leukemia during pregnancy. We prospect that fetal cells, including hepatocytes and endothelial and hematopoietic cells, might regulate fetal liver HSC expansion. Components from vascular endothelial cells and bones might also modulate the lodging of fetal liver HSCs into the bone marrow. The current review holds great potential to deeply understand the molecular regulations of HSCs in the fetal liver and bone marrow in mammals, which will be helpful to efficiently expand HSCs in vitro.

Induction of Adipocyte Differentiation and Reduction of Hematopoesis-Supporting Activity of Mouse Stromal Cells by Suppression of Transcription Factor GATA-2.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1392-1392
Author(s):  
Yoko Okitsu ◽  
Hideo Harigae ◽  
Masanori Seki ◽  
Toru Fujiwara ◽  
Shinichiro Takahashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Colony Formation ◽  
Adipocyte Differentiation ◽  
Fetal Liver ◽  
Expression Level ◽  
Hematopoietic Stem

Abstract (Introduction) Aplastic anemia (AA) is characterized by peripheral pancytopenia and fatty bone marrow. An immunological attack to hematopoietic stem cells has been thought to be responsible for the development of the disease. Previously, we reported the expression of transcription factor GATA-2 is significantly decreased in CD34 positive cells in AA. Together with the phenotypes of hematopoietic stem cells in GATA-2 hetero-knockout mice, GATA-2 down-regulation may play a role in the reduction of a stem cell pool observed in AA. On the other hand, GATA-2 has been shown to be essential for the maintenance of immaturity of preadipocytes. If a pathological immune response in AA decreases the level of GATA-2 expression in not only hematopoietic stem cells but also stromal preadipocytes, it may accelerate the maturation of preadipocytes, leading to the formation of fatty bone marrow. To explore this possibility, the phenotypic change of stromal preadipocytes by suppression of GATA-2 was examined in this study. (Method) The GATA-2 expression level was suppressed by using siRNA for GATA-2 in mouse stromal preadipocyte cell lines, TBR9 and TBR343. After the treatment with siRNA, the adipocyte differentiation was induced by the incubation with insulin and dexamethasone for 7days. Then, the maturation level was examined by oil drops formation judged by oil red staining, and by the expression level of adipcin and PPAR-γ mRNA. Supporting activity of hematopoietic colony formation was also evaluated by using mouse fetal liver cells after siRNA treatment. (Results) By using designed siRNA, the GATA-2 expression was suppressed to 30% of control, whereas the expression level of GATA-3, which is co-expressed in preadipocytes, was unchanged. When GATA-2 was suppressed by siRNA, the oil drop formation and adipocyte-specific gene expression was significantly accelerated in both of stromal cells. Furthermore, the number of fetal liver hematopoetic colonies was significantly decreased by suppression of GATA-2, suggesting that GATA-2 down-regulation in stromal preadipocytes results in not only the acceleration of the maturation but also the reduced supporting activity of hematopoietic colony formation (Conclusion) These results suggest that suppression of GATA-2 in hematopoietic tissues induces the characteristic features of AA, i.e., decreased the number of hematopoietic stem cells and increased number of mature adipocytes.

Engraftment of Human Mesenchymal Stem Cells upon Transplantation into Newborn Rag2−/−gc−/− Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1652-1652
Author(s):  
Patrick Ziegler ◽  
Steffen Boettcher ◽  
Hildegard Keppeler ◽  
Bettina Kirchner ◽  
Markus G. Manz
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Human Mesenchymal Stem Cells ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Gene Transcripts

Abstract We recently demonstrated human T cell, B cell, dendritic cell, and natural interferon producing cell development and consecutive formation of primary and secondary lymphoid organs in Rag2−/−gc−/− mice, transplanted as newborns intra-hepatically (i.h.) with human CD34+ cord blood cells (Traggiai et al., Science 2004). Although these mice support high levels of human cell engraftment and continuous T and B cell formation as well as CD34+ cell maintenance in bone marrow over at least six month, the frequency of secondary recipient reconstituting human hematopoietic stem and progenitor cells within the CD34+ pool declines over time. Also, although some human immune responses are detectable upon vaccination with tetanus toxoid, or infection with human lymphotropic viruses such as EBV and HIV, these responses are somewhat weak compared to primary human responses, and are inconsistent in frequency. Thus, some factors sustaining human hematopoietic stem cells in bone marrow and immune responses in lymphoid tissues are either missing in the mouse environment, or are not cross-reactive on human cells. Human mesenchymal stem cells (MSCs) replicate as undifferentiated cells and are capable to differentiate to multiple mesenchymal tissues such as bone, cartilage, fat, muscle, tendon, as well as marrow and lymphoid organ stroma cells, at least in vitro (e.g. Pittenger et al., Science 1999). Moreover, it was shown that MSCs maintain CD34+ cells to some extend in vitro, and engraft at low frequency upon transplantation into adult immunodeficient mice or fetal sheep as detected by gene transcripts. We thus postulated that co-transplantation of cord blood CD34+ cells and MSCs into newborn mice might lead to engraftment of both cell types, and to provision of factors supporting CD34+ maintenance and immune system function. MSCs were isolated and expanded by plastic adherence in IMDM, supplemented with FCS and cortisone (first 3 weeks) from adult bone marrow, cord blood, and umbilical vein. To test their potential to support hemato-lymphopoiesis, MSCs were analyzed for human hemato-lymphotropic cytokine transcription and production by RT-PCR and ELISA, respectively. MSCs from all sources expressed gene-transcripts for IL-6, IL-7, IL-11, IL-15, SCF, TPO, FLT3L, M-CSF, GM-CSF, LIF, and SDF-1. Consistently, respective cytokines were detected in supernatants at the following, declining levels (pg/ml): IL-6 (10000-10E6) > SDF-1 > IL-11 > M-CSF > IL-7 > LIF > SCF > GM-CSF (0–450), while FLT3L and TPO were not detectable by ELISA. Upon i.h. transplantation of same passage MSCs (1X10E6) into sublethally irradiated (2x2 Gy) newborn Rag2−/−gc−/− mice, 2-week engraftment was demonstrated by species specific b2m-RT-PCR in thymus, spleen, lung, liver and heart in n=7 and additionally in thymus in n=3 out of 13 animals analyzed. Equally, GFP-RNA transcripts were detectable in the thymus for up to 6 weeks, the longest time followed, upon co-transplantation of same source CD34+ cells and retrovirally GFP-transduced MSCs in n=2 out of 4 animals. Further engraftment analysis of ongoing experiments will be presented. Overall, these results demonstrate that human MSC produce hemato-lymphoid cytokines and engraft in newborn transplanted Rag2−/−gc−/− mice, at least at early time-points analyzed. This model thus might allow studying hematopoietic cell and MSC-derived cell interaction, and might serve as a testing system for MSC delivered gene therapy in vivo.

Partial Characterization and In Vitro Expansion of Putative CLL Precursor/Stem Cells Which Are Dependent on Bone Marrow Microenvironment for Survival

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2433-2433
Author(s):  
Medhat Shehata ◽  
Rainer Hubmann ◽  
Martin Hilgarth ◽  
Susanne Schnabl ◽  
Dita Demirtas ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Healthy Persons

Abstract Abstract 2433 Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of B lymphocytes which typically express CD19 and CD5. The disease remains incurable and recurrence often occurs after current standard therapies due to residual disease or probably due to the presence of therapy-resistant CLL precursors. Based on the growing evidence for the existence of leukemia stem cells, this study was designed to search for putative CLL precursors/stem cells based on the co-expression of CLL cell markers (CD19/CD5) with the hematopoietic stem cell marker (CD34). Forty seven CLL patients and 17 healthy persons were enrolled in the study. Twenty four patients had no previous treatment and 23 had pre-therapy. Twenty two patients were in Binet stage C and 25 patients in B. Twenty two patients had unmutated and 18 mutated IgVH gene (7: ND). Cytogenetic analysis by FISH showed that 14 patients had del 13q, 8 had del 11q, 4 had del 17p and 9 had trisomy 12. Peripheral blood and bone marrow mononuclear cells were subjected to multi-colour FACS analysis using anti-human antibodies against CD34, CD19 and CD5 surface antigens. The results revealed the presence of triple positive CD34+/CD19+/CD5+ cells in CLL samples (mean 0.13%; range 0.01–0.41) and in healthy donors (0.31%; range 0.02–0.6) within the CD19+ B cells. However, due to the high leukocyte count in CLL patients, the absolute number of these cells was significantly higher in CLL samples (mean: 78.7; range 2.5–295 cells /μL blood) compared to healthy persons (mean: 0.45: range 0.04–2.5 cells/μl)(p<0,001). These triple positive “putative CLL stem cells” (PCLLSC) co-express CD133 (67%), CD38 (87%), CD127 (52%), CD10 (49%), CD20 (61%), CD23 (96%), CD44 (98%) and CD49d (74%). FISH analysis on 4 patients with documented chromosomal abnormalities detected the corresponding chromosomal aberrations of the mature clone in the sorted CD34+/CD5+/CD19+ and/or CD34+/CD19-/CD5- cells but not in the CD3+ T cells. Multiplex RT-PCR analysis using IgVH family specific primer sets confirmed the clonality of these cells. Morphologically, PCLLSC appeared larger than lymphocytes with narrow cytoplasm and showed polarity and motility in co-culture with human bone marrow stromal cells. Using our co-culture microenvironment model (Shehata et al, Blood 2010), sorted cell fractions (A: CD34+/19+/5+, B: CD34+/19-/5- or C: CD34-/CD19+/5+) from 4 patients were co-cultured with primary autologous human stromal cells. PCLLSC could be expanded in the co-culture to more than 90% purity from fraction A and B but not from fraction C. These cells remained in close contact or migrated through the stromal cells. PCLLSC required the contact with stromal cells for survival and died within 1–3 days in suspension culture suggesting their dependence on bone marrow microenvironment or stem cell niches. RT-PCR demonstrated that these cells belong to the established CLL clone. They also eexpress Pax5, IL-7R, Notch1, Notch2 and PTEN mRNA which are known to play a key role in the early stages of B cells development and might be relevant to the early development of the malignant clone in CLL. Using NOD/SCID/IL2R-gamma-null (NOG) xenogeneic mouse system we co-transplanted CLL cells from 3 patients (5 million PBMC/mouse) together with autologous bone marrow stromal cells (Ratio: 10:1). The percentage of PCLLSC in the transplanted PBMC was 0.18% (range 0.06–0.34%). Using human-specific antibodies, human CD45+ cells were detected in peripharal blood of the mice (mean 0.9 % range 0.47–1.63%) after 2 months of transplantation. More than 90% of the human cells were positive for CD45 and CD5. Among this population, 26% (range 15–35%) of the cells co-expressed CD45, CD19, CD5 and CD34 and thus correspond to the PCLLSC. In conclusion, our data suggest the existence of putative CLL precursors/stem cells which reside within the CD34+ hematopoietic stem cell compartment and carry the chromosomal aberrations of the established CLL clone. These cells could be expanded in vitro in a bone marrow stroma-dependent manner and could be engrafted and significantly enriched in vivo in NOG xenotransplant system. Further characterization and selective targeting and eradication of these cells may pave the way for designing curative therapeutic strategies for CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In VitroLarge Scale Production of Human Mature Red Blood Cells from Hematopoietic Stem Cells by Coculturing with Human Fetal Liver Stromal Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Jiafei Xi ◽  
Yanhua Li ◽  
Ruoyong Wang ◽  
Yunfang Wang ◽  
Xue Nan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Red Blood Cells ◽  
Stromal Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Hematopoietic Stem

In vitromodels of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 109-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitiveβ-globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.

scholarly journals Lymphoid reconstitution of the human fetal thymus in SCID mice with CD34+ precursor cells.

1991 ◽  
Vol 174 (5) ◽  
pp. 1283-1286 ◽  
Author(s):  
B Péault ◽  
I L Weissman ◽  
C Baum ◽  
J M McCune ◽  
A Tsukamoto
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Precursor Cell ◽  
Scid Mice ◽  
Hematopoietic Stem ◽  
Fetal Thymus

The search for human hematopoietic stem cells has been hampered by the lack of appropriate assay systems. Demonstration of the ability of precursor cell candidates to give rise to T cells is of significant difficulty since dissociated in vitro cultured thymus stroma cells lose their ability to sustain thymocyte maturation. To define further the differentiative capacities of the rare human fetal liver and bone marrow cells that express the CD34 surface antigen and exhibit in vitro myeloid and pre-B cell activities, we have microinjected them into HLA-mismatched fetal thymus fragments, partially depleted of hematopoietic cells by low temperature culture. In vitro colonized thymuses have then been allowed to develop upon engraftment into immunodeficient SCID mice. Using this modification of the SCID-hu system, we show that low numbers of fetal CD34+ progenitor cells can repopulate the lymphoid compartment in the human thymus.

Kinetics of hematopoietic stem cells and supportive activities of stromal cells in a three-dimensional bone marrow culture system

2015 ◽  
Vol 33 (5-6) ◽  
pp. 347-355 ◽  
Author(s):  
Tomonori Harada ◽  
Yukio Hirabayashi ◽  
Yoshihiro Hatta ◽  
Isao Tsuboi ◽  
Wilhelm Robert Glomm ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Culture System ◽  
Three Dimensional ◽  
Bone Marrow Culture ◽  
Hematopoietic Stem ◽  
Kinetics Of

scholarly journals Presence of mixed colony-forming cells in long-term hamster bone marrow suspension cultures: response to pokeweed spleen conditioned medium

Blood ◽  
1982 ◽  
Vol 60 (2) ◽  
pp. 495-502
Author(s):  
CE Eastment ◽  
FW Ruscetti ◽  
E Denholm ◽  
I Katznelson ◽  
E Arnold ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Conditioned Medium ◽  
In Vitro Study ◽  
Suspension Cultures ◽  
Rapid Expansion ◽  
Pokeweed Mitogen ◽  
Hematopoietic Stem

In contrast to the murine system, long-term hamster bone marrow suspension cultures maintain proliferation of both pluripotent and committed stem cells in the absence of an adherent layer and without addition of exogenous factors, such as hydrocortisone. Addition of pokeweed-mitogen-stimulated hamster spleen conditioned medium (SCM) to these long-term suspension cultures produces an increase in the number of mixed colonies assayed in soft-agar, These mixed colonies, which contained four cell lineages--granulocytic, erythroid, megakaryocytic, and macrophage--could be generated from cells grown in suspension for over 6 mo. Addition of SCM also induces an initial rapid expansion of the myeloid compartment, and this expansion results in 70% of the cells being terminally differentiated granulocytes. In contrast, addition of SCM to hamster bone marrow cultures containing both adherent cells and hematopoietic stem cells produced no change in the number of mixed colonies generated in the culture. This system allows the in vitro study of the process of stem cell proliferation and differentiation and also provides a means to examine the relationship of adherent and supernatant bone marrow populations.

Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem and Progenitor Cells From Acid Sphingomyelinase-Deficient Mice: Implications for Niemann-Pick Disease Gene Therapy and the Development of Improved Stem Cell Gene Transfer Procedures

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 80-86 ◽  
Author(s):  
Shai Erlich ◽  
Silvia R.P. Miranda ◽  
Jan W.M. Visser ◽  
Arie Dagan ◽  
Shimon Gatt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Acid Sphingomyelinase ◽  
Hematopoietic Stem

Abstract The general utility of a novel, fluorescence-based procedure for assessing gene transfer and expression has been demonstrated using hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic cells were isolated from the bone marrow or fetal livers of acid sphingomyelinase–deficient mice, and retrovirally transduced with amphotropic or ecotropic vectors encoding a normal acid sphingomyelinase (ASM) cDNA. Anti–c-Kit antibodies were then used to label stem- and progenitor-enriched cell populations, and the Bodipy fluorescence was analyzed in each group after incubation with a Bodipy-conjugated sphingomyelin. Only cells expressing the functional ASM (ie, transduced) could degrade the sphingomyelin, thereby reducing their Bodipy fluorescence as compared with nontransduced cells. The usefulness of this procedure for the in vitro assessment of gene transfer into hematopoietic stem cells was evaluated, as well as its ability to provide an enrichment of transduced stem cells in vivo. To show the value of this method for in vitro analysis, the effects of retroviral transduction using ecotropic versus amphotropic vectors, various growth factor combinations, and adult bone marrow versus fetal liver stem cells were assessed. The results of these studies confirmed the fact that ecotropic vectors were much more efficient at transducing murine stem cells than amphotropic vectors, and that among the three most commonly used growth factors (stem cell factor [SCF] and interleukins 3 and 6 [IL-3 and IL-6]), SCF had the most significant effect on the transduction of stem cells, whereas IL-6 had the most significant effect on progenitor cells. In addition, it was determined that fetal liver stem cells were only approximately twofold more “transducible” than stem cells from adult bone marrow. Transplantation of Bodipy-selected bone marrow cells into lethally irradiated mice showed that the number of spleen colony-forming units that were positive for the retroviral vector (as determined by polymerase chain reaction) was 76%, as compared with 32% in animals that were transplanted with cells that were nonselected. The methods described within this manuscript are particularly useful for evaluating hematopoietic stem cell gene transfer in vivo because the marker gene used in the procedure (ASM) encodes a naturally occurring mammalian enzyme that has no known adverse effects, and the fluorescent compound used for selection (Bodipy sphingomyelin) is removed from the cells before transplantation.

In Vivo Generation of Engraftable Murine Hematopoietic Stem Cells from Induced Pluripotent Stem Cells through Hemogenic Endothelium

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3866-3866
Author(s):  
Masao Tsukada ◽  
Satoshi Yamazaki ◽  
Yasunori Ota ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Teratoma Formation

Abstract Introduction Generation of engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) has long been thought an ultimate goal in the field of hematology. Numerous in vitro differentiation protocols, including trans-differentiation and forward programming approaches, have been reported but have so far failed to generate fully functional HSCs. We have previously demonstrated proof-of-concept for the in vivo generation of fully functional HSCs from induced PSCs (iPSCs) through teratoma formation (Suzuki et al., 2013). However, this method is time-consuming (taking over two months), HSCs are generated at low frequencies, and additionally require co-injection on OP9 stromal cells and SCF/TPO cytokines. Here, we present optimization of in vivo HSC generation via teratoma formation for faster, higher-efficiency HSC generation and without co-injection of stromal cells or cytokines. Results First, we screened reported in vitro trans-differentiation and forward programming strategies for their ability to generate HSCs in vivo within the teratoma assay. We tested iPSCs transduced with the following dox-inducible TF overexpression vectors: (1) Gfi1b, cFOS and Gata2 (GFG), which induce hemogenic endothelial-like cells from fibroblast (Pereira et al.,2013); (2) Erg, HoxA9 and Rora (EAR), which induce short-term hematopoietic stem/progenitor cell (HSPC) formation during embryoid body differentiation (Doulatov et,al., 2013); and (3) Foxc1, which is highly expressed the CAR cells, a critical cell type for HSC maintenance (Oomatsu et al.,2014). We injected iPSCs into recipient mice, without co-injection of stromal cells or cytokines, and induced TF expression after teratoma formation by dox administration. After four weeks, GFG-derived teratomas contained large numbers of endothelial-like and epithelial-like cells, and importantly GFG-derived hematopoietic cells could also be detected. EAR-teratomas also generated hematopoietic cells, although at lower frequencies. By contrast, hematopoietic cells were not detected in control teratomas or Foxc1-teratomas. Through use of iPSCs generated from Runx1-EGFP mice (Ng et al. 2010), and CUBIC 3D imaging technology (Susaki et al. 2014), we were further able to demonstrate that GFG-derived hematopoietic cells were generated through a haemogenic endothelium precursor. Next, we assessed whether HSPC-deficient recipient mice would allow greater expansion of teratoma-derived HSCs. This was achieved by inducing c-kit deletion within the hematopoietic compartment of recipient mice (Kimura et al., 2011) and resulted in a ten-fold increase in the peripheral blood frequency of iPSC-derived hematopoietic cells. We further confirmed similar increases in iPSC-derived bone marrow cells, and in vivo HSC expansion, through bone marrow transplantation assays. Finally, we have been able to shorten the HSC generation time in this assay by five weeks through use of transplantable teratomas, rather than iPSCs. Conclusions We have demonstrated that GFG-iPSCs induce HSC generation within teratomas, via a hemogenic endothelium precursor, and that use of HSPC-deficient recipient mice further promotes expansion of teratoma-derived HSCs. These modifications now allow us to generate engraftable HSCs without co-injection of stromal cells or cytokines. Additionally, use of transplantable teratomas reduced HSC generation times as compared with the conventional assay. These findings suggest that our in vivo system provides a promising strategy to generate engraftable HSCs from iPSCs. Disclosures No relevant conflicts of interest to declare.

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