Comprehensive Clonal Mapping of Hematopoiesis in Vivo in Humans By Retroviral Vector Insertional Barcoding

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5-5
Author(s):  
Luca Biasco ◽  
Serena Scala ◽  
Francesca Dionisio ◽  
Andrea Calabria ◽  
Luca Basso Ricci ◽  
...  
Keyword(s):  
Detailed Analysis ◽  
Graphical Models ◽  
Integration Site ◽  
Cell Types ◽  
Retroviral Vector ◽  
Lymphoid Cells ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSC) are endowed with the unique role of generating an adequate and efficient pool of blood cells throughout human life. Data derived from clonal tracking of HSC activity and hematopoietic dynamics directly in vivo in humans would be of paramount importance for the design of therapies for hematological disorders and cancers. Our gene therapy (GT) clinical trials for adenosine deaminase (ADA) deficient-SCID and Wiskott-Aldrich Syndrome (WAS) based on the infusion of genetically engineered HSC, constitute unique clinical settings where each vector-marked progenitors and its blood cell progeny is traceable being univocally barcoded by a vector integration site (IS). To study early dynamics of hematopoietic reconstitution in humans, we collected by LAM-PCR + Illumina-Miseq sequencing 14.807.407 sequence reads corresponding to 71.981 IS tagging clones belonging to 13 different cell types purified from the bone marrow and the peripheral blood of 4 WAS patients up to 36 months after GT. We firstly identified and quantified identical IS shared among CD34+ progenitors, and mature Myeloid/Lymphoid cells as marker of the real-time clonal output of individual vector-marked HSC clones in vivo. We unraveled the timing of short, intermediate and long term HSC output showing that CD34+ clones active at 3-6 months after GT are not detectable at later follow up. By unsupervised clustering of IS similarities among lineages we unveiled diverse input of HSPC clonal differentiation towards lymphoid, myeloid and megakaryo-erythroid cells and found that NK cells have a distinct relationship with HSPC as compared to T and B cells. We also profiled the level of HSPC output overtime showing that early reconstitution is markedly skewed towards myeloid production. Importantly, clonogenic progenitors generated in vitro from ex vivo purified CD34+ patients’ cells, showed a IS profile coherent with that of freshly purified BM and PB cell types from the same time-point. We also studied population clonal entropy through 7 different diversity indexes and uncovered that progenitor output occurs in distinct waves during the first 6-9 months after transplantation reaching a “homeostatic equilibrium” only by 12 months after GT. At steady state we estimated by mark-recapture mathematical approaches that 1900-7000 transduced HSC clones were stably contributing to the progenitors repertoire for up to 3 years after infusion of gene corrected CD34+ cells. To evaluate the long-term preservation of activity by transplanted HSC we exploited data derived from the IS-based tracking of 4.845 clones in ADA-SCID patients performed for up to 6 years after GT. We showed that identical IS are consistently detected at multiple lineages level even several years after GT. Strikingly, by semi-quantitative PCRs on specific vector-genome junctions we tracked a fluctuating but consistent output of marked HSC over a period of 5 years without the manifestation of clonal quiescence phases. Additionally, since the gamma-retroviral vector used in ADA-SCID HSC-GT trial is able to transduce only actively replicating cells, we provided the first evidence that in vitro activated HSC, “awaken” from dormancy, can still, once infused, retain in vivo long-term activity in humans. We exploited IS similarities among the lineages for both WAS and ADA-SCID datasets to reconstruct the hematopoietic hierarchy by combining conditional probability distributions and static/dynamic graphical models of dependencies. Notably, preliminary data unveiled a link between myeloid progenitors and mature lymphoid cells that supports the recently suggested model of hematopoiesis based on a delayed branching of myeloid and lymphoid lineages. Further mathematical models are being applied to specifically study population dynamics and single HSPC contribution to hematopoiesis including stochastic models of neutral clonal drift. More detailed analysis are also being performed on IS collected from 7 distinct CD34+ subtypes isolated from GT patients and FACS sorted according to the most recent markers of HSPC differentiation. Overall our work constitute the first molecular tracking of individual hematopoietic clones in humans providing an unprecedented detailed analysis of HSC activity and dynamics in vivo. The information gathered will be crucial for the design of therapeutic approaches for a broad spectrum of hematological diseases and tumors. Disclosures Neduva: GSK: Employment. Dow:GSK: Employment.

scholarly journals Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil- treated murine marrow

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2310-2320 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Functional Characterization ◽  
Significant Proportion ◽  
Cell Types ◽  
Hematopoietic Stem ◽  
Clonogenic Cell

Lymphomyeloid stem cells from the bone marrow of C57BL/6 mice treated with 5-fluorouracil (5-FU) were characterized with respect to 12 parameters using fluorescence-activated cell sorting and a competitive long-term repopulation assay. Stem cells were larger than lymphocytes and exhibited side light-scatter characteristic of blast cells. Most expressed low levels of Thy-1.2, high levels of Sca-1 (Ly6-A/E), H-2Kb, and AA4.1 antigens and stained brightly with rhodamine-123. Significantly, most long-term repopulating cells also expressed CD4, some at high density. In addition, a significant proportion displayed low to medium levels of the “lineage-specific” markers CD45R (B220), Gr- 1, and TER-119. A simple and rapid multiparameter sorting procedure enriched the stem cells 100-fold and substantially removed most other clonogenic cell types, including day 12 spleen colony-forming cells. Cells able to generate cobblestone colonies on stromal cells in vitro were co-enriched. Lethally irradiated mice transplanted with limiting numbers of the sorted stem cells did not survive unless cotransplanted with “compromised” marrow cells prepared by prior serial transplantation and shown to be depleted of long-term repopulating activity. A significant number of recipients transplanted with 25 to 100 sorted cells contained donor-derived B and T lymphocytes and granulocytes in their peripheral blood for at least 6 months. Limiting dilution analysis in vivo indicated that the frequency of competitive long-term repopulating units (CRU) in the sorted population was at least 1 in 60 cells. The calculated frequency of CRU was largely independent of the time of recipient analysis between 10 and 52 weeks, indicating that highly enriched stem cells can be recruited relatively early in certain transplant settings. This simple enrichment and assay strategy for repopulating hematopoietic stem cells should facilitate further analysis of their regulation in vivo.

scholarly journals Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil- treated murine marrow

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2310-2320 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Functional Characterization ◽  
Significant Proportion ◽  
Cell Types ◽  
Hematopoietic Stem ◽  
Clonogenic Cell

Abstract Lymphomyeloid stem cells from the bone marrow of C57BL/6 mice treated with 5-fluorouracil (5-FU) were characterized with respect to 12 parameters using fluorescence-activated cell sorting and a competitive long-term repopulation assay. Stem cells were larger than lymphocytes and exhibited side light-scatter characteristic of blast cells. Most expressed low levels of Thy-1.2, high levels of Sca-1 (Ly6-A/E), H-2Kb, and AA4.1 antigens and stained brightly with rhodamine-123. Significantly, most long-term repopulating cells also expressed CD4, some at high density. In addition, a significant proportion displayed low to medium levels of the “lineage-specific” markers CD45R (B220), Gr- 1, and TER-119. A simple and rapid multiparameter sorting procedure enriched the stem cells 100-fold and substantially removed most other clonogenic cell types, including day 12 spleen colony-forming cells. Cells able to generate cobblestone colonies on stromal cells in vitro were co-enriched. Lethally irradiated mice transplanted with limiting numbers of the sorted stem cells did not survive unless cotransplanted with “compromised” marrow cells prepared by prior serial transplantation and shown to be depleted of long-term repopulating activity. A significant number of recipients transplanted with 25 to 100 sorted cells contained donor-derived B and T lymphocytes and granulocytes in their peripheral blood for at least 6 months. Limiting dilution analysis in vivo indicated that the frequency of competitive long-term repopulating units (CRU) in the sorted population was at least 1 in 60 cells. The calculated frequency of CRU was largely independent of the time of recipient analysis between 10 and 52 weeks, indicating that highly enriched stem cells can be recruited relatively early in certain transplant settings. This simple enrichment and assay strategy for repopulating hematopoietic stem cells should facilitate further analysis of their regulation in vivo.

scholarly journals Trajectory of chemical cocktail-induced neutrophil reprogramming

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Yi Zhou ◽  
Chuijin Wei ◽  
Shumin Xiong ◽  
Liaoliao Dong ◽  
Zhu Chen ◽  
...  
Keyword(s):  
Cell Types ◽  
Lymphoid Cells ◽  
Great Promise ◽  
Target Cells ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
First Time

AbstractHematopoietic reprogramming holds great promise for generating functional target cells and provides new angle for understanding hematopoiesis. We reported before for the first time that diverse differentiated hematopoietic cell lineages could be reprogrammed back into hematopoietic stem/progenitor cell-like cells by chemical cocktail. However, the exact cell types of induced cells and reprogramming trajectory remain elusive. Here, based on genetic tracing method CellTagging and single-cell RNA sequencing, it is found that neutrophils could be reprogrammed into multipotent progenitors, which acquire multi-differentiation potential both in vitro and in vivo, including into lymphoid cells. Construction of trajectory map of the reprogramming procession shows that mature neutrophils follow their canonical developmental route reversely into immature ones, premature ones, granulocyte/monocyte progenitors, common myeloid progenitors, and then the terminal cells, which is stage by stage or skips intermediate stages. Collectively, this study provides a precise dissection of hematopoietic reprogramming procession and sheds light on chemical cocktail-induction of hematopoietic stem cells.

scholarly journals Identification of properties that can distinguish primitive populations of stromal-cell-responsive lympho-myeloid cells from cells that are stromal-cell-responsive but lymphoid-restricted and cells that have lympho-myeloid potential but are also capable of competitively repopulating myeloablated recipients

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1639-1648 ◽  
Author(s):  
ME Lemieux ◽  
CJ Eaves
Keyword(s):  
Stromal Cell ◽  
Adult Mouse ◽  
Cell Types ◽  
Second Phase ◽  
Hematopoietic Stem ◽  
Stem Cell Regulation ◽  
B Lineage

Abstract We have recently described a modification of the long-term culture- initiating cell (LTC-IC) assay that allows the identification and quantitation of a subset of murine LTC-IC that are able to generate both myeloid and lymphoid progeny in vitro. These “LTC-ICML” are inseparable from either standard LTC-IC or cells with long-term in vivo lympho-myeloid repopulating potential (competitive repopulating units, or CRU) present in normal adult mouse marrow but are detected at 15- to 30-fold lower frequencies. To determine whether at least part of this discrepancy is due to a reduced sensitivity of the LTC-ICML assay, a number of strategies to try to improve the original protocol were tested. Prolongation of the second phase of the assay (when the cells are maintained under lymphoid LTC conditions) from 7 to 10 days and inclusion of Flt3 ligand (FL) in the assay used to detect the production of B-lineage cells (CFU-pre-B) increased the average yield of CFU-pre-B per LTC-ICML by a factor of 3- to 20-fold; however, neither of these manipulations increased the frequency of LTC-ICML detected. Similarly, the use of S17 fibroblasts as feeders also did not improve the sensitivity of the LTC-ICML assay. On the other hand, approximately threefold more LTC-ICML could be detected when the initial phase of the assay (when the cells are maintained under myeloid LTC conditions) was shortened from 4 weeks to 1 week. In addition, this latter modification showed the existence in adult mouse marrow of a previously unrecognized, very early B220- stage of lymphoid development characterized by a relative resistance to both 5-fluorouracil and hydrocortisone. The discovery of such cells is of significant interest; however, their presence in adult mouse marrow in numbers comparable to those of both LTC-ICML and myeloid-restricted LTC-IC imposes severe restrictions on the use of the shorter LTC-ICML assay which offset its increased sensitivity. Interestingly, the present studies also show that both LTC-IC and LTC-ICML numbers are significantly better maintained (approximately twofold to sixfold) in myeloid LTC than are CRU. This differential maintenance of LTC-IC and CRU in vitro may be related to the fact that the detection of each of these two functionally defined progenitors involves the use of different ligand receptor systems. Alternatively, it is possible that at least some LTC- IC and CRU may represent developmentally distinct cell types. Taken together, these findings suggest a model of hematopoietic stem cell regulation in which retention of totipotentiality and maintenance of responsiveness to specific regulators may not be tightly linked.

scholarly journals Identification of properties that can distinguish primitive populations of stromal-cell-responsive lympho-myeloid cells from cells that are stromal-cell-responsive but lymphoid-restricted and cells that have lympho-myeloid potential but are also capable of competitively repopulating myeloablated recipients

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1639-1648 ◽  
Author(s):  
ME Lemieux ◽  
CJ Eaves
Keyword(s):  
Stromal Cell ◽  
Adult Mouse ◽  
Cell Types ◽  
Second Phase ◽  
Hematopoietic Stem ◽  
Stem Cell Regulation ◽  
B Lineage

We have recently described a modification of the long-term culture- initiating cell (LTC-IC) assay that allows the identification and quantitation of a subset of murine LTC-IC that are able to generate both myeloid and lymphoid progeny in vitro. These “LTC-ICML” are inseparable from either standard LTC-IC or cells with long-term in vivo lympho-myeloid repopulating potential (competitive repopulating units, or CRU) present in normal adult mouse marrow but are detected at 15- to 30-fold lower frequencies. To determine whether at least part of this discrepancy is due to a reduced sensitivity of the LTC-ICML assay, a number of strategies to try to improve the original protocol were tested. Prolongation of the second phase of the assay (when the cells are maintained under lymphoid LTC conditions) from 7 to 10 days and inclusion of Flt3 ligand (FL) in the assay used to detect the production of B-lineage cells (CFU-pre-B) increased the average yield of CFU-pre-B per LTC-ICML by a factor of 3- to 20-fold; however, neither of these manipulations increased the frequency of LTC-ICML detected. Similarly, the use of S17 fibroblasts as feeders also did not improve the sensitivity of the LTC-ICML assay. On the other hand, approximately threefold more LTC-ICML could be detected when the initial phase of the assay (when the cells are maintained under myeloid LTC conditions) was shortened from 4 weeks to 1 week. In addition, this latter modification showed the existence in adult mouse marrow of a previously unrecognized, very early B220- stage of lymphoid development characterized by a relative resistance to both 5-fluorouracil and hydrocortisone. The discovery of such cells is of significant interest; however, their presence in adult mouse marrow in numbers comparable to those of both LTC-ICML and myeloid-restricted LTC-IC imposes severe restrictions on the use of the shorter LTC-ICML assay which offset its increased sensitivity. Interestingly, the present studies also show that both LTC-IC and LTC-ICML numbers are significantly better maintained (approximately twofold to sixfold) in myeloid LTC than are CRU. This differential maintenance of LTC-IC and CRU in vitro may be related to the fact that the detection of each of these two functionally defined progenitors involves the use of different ligand receptor systems. Alternatively, it is possible that at least some LTC- IC and CRU may represent developmentally distinct cell types. Taken together, these findings suggest a model of hematopoietic stem cell regulation in which retention of totipotentiality and maintenance of responsiveness to specific regulators may not be tightly linked.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 930-939 ◽  
Author(s):  
SJ Szilvassy ◽  
PM Lansdorp ◽  
RK Humphries ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Simple Procedure ◽  
Hematopoietic Cells ◽  
Stem Cell Population ◽  
Proliferative Potential ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male “test” cells and 1 to 2 x 10(5) “compromised” female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5- FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

scholarly journals B-1 lymphoid cells develop independently of Notch signaling during mouse embryonic development

2020 ◽  
Author(s):  
Nathalia Azevedo ◽  
Elisa Bertesago ◽  
Ismail Ismailoglu ◽  
Michael Kyba ◽  
Michihiro Kobayashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Development ◽  
Blood Cell ◽  
Notch Signaling ◽  
Embryonic Stem ◽  
Cell Types ◽  
Lymphoid Cells ◽  
Lineage Specification ◽  
Hematopoietic Stem

AbstractThe in vitro generation from pluripotent stem cells (PSCs) of different blood cell types, in particular those that are not replenished by hematopoietic stem cells (HSCs) like fetal-derived tissue-resident macrophages and innate-like lymphocytes, is of a particular interest. In order to succeed in this endeavor, a thorough understanding of the pathway interplay promoting lineage specification for the different blood cell types is needed. Notch signaling is essential for the HSC generation and their derivatives, but its requirement for tissue-resident immune cells is unknown. Using mouse embryonic stem cells (mESCs) to recapitulate murine embryonic development, we have studied the requirement for Notch signaling during the earliest B-lymphopoiesis and found that Rbpj-deficient mESCs are able to generate B-1 cells. Their Notch-independence was confirmed in ex vivo experiments using Rbpj-deficient embryos. In addition, we found that upregulation of Notch signaling was needed for the emergence of B-2 lymphoid cells. Taken together, these findings indicate that control of Notch signaling dosage is critical for the different B-cell lineage specification and provides pivotal information for their in vitro generation from PSCs for therapeutic applications.

scholarly journals Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4773-4777 ◽  
Author(s):  
Hal E. Broxmeyer ◽  
Man-Ryul Lee ◽  
Giao Hangoc ◽  
Scott Cooper ◽  
Nutan Prasain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1748-1755 ◽  
Author(s):  
David Bryder ◽  
Sten E. W. Jacobsen
Keyword(s):  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Calf Serum ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Divisions ◽  
Stem Cell Divisions

Abstract Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

Sign in / Sign up

Export Citation Format

Share Document