scholarly journals Asymmetric Division and Lineage Commitment at the Level of Hematopoietic Stem Cells

2004 ◽  
Vol 199 (3) ◽  
pp. 295-302 ◽  
Author(s):  
Hina Takano ◽  
Hideo Ema ◽  
Kazuhiro Sudo ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
Asymmetric Division ◽  
Lineage Commitment ◽  
Mouse Bone Marrow ◽  
Differentiation Potential ◽  
Hematopoietic Stem

How hematopoietic stem cells (HSCs) commit to a particular lineage is unclear. A high degree of HSC purification enabled us to address this issue at the clonal level. Single-cell transplantation studies revealed that 40% of the CD34−/low, c-Kit+, Sca-1+, and lineage marker− (CD34−KSL) cells in adult mouse bone marrow were able, as individual cells, to reconstitute myeloid and B- and T-lymphoid lineages over the long-term. Single-cell culture showed that >40% of CD34−KSL cells could form neutrophil (n)/macrophage (m)/erythroblast (E)/megakaryocyte (M) (nmEM) colonies. Assuming that a substantial portion of long-term repopulating cells can be detected as nmEM cells within this population, we compared differentiation potentials between individual pairs of daughter and granddaughter cells derived in vitro from single nmEM cells. One of the two daughter or granddaughter cells remained an nmEM cell. The other showed a variety of combinations of differentiation potential. In particular, an nmEM cell directly gave rise, after one cell division, to progenitor cells committed to nm, EM, or M lineages. The probability of asymmetric division of nmEM cells depended on the cytokines used. These data strongly suggest that lineage commitment takes place asymmetrically at the level of HSCs under the influence of external factors.

Evidence for a Novel Blood Lineage Commitment Pathway from Lympho-Myeloid Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 802-802 ◽  
Author(s):  
Sten Eirik W. Jacobsen ◽  
Robert Mansson ◽  
Anne Hultquist ◽  
Mikael Sigvardsson ◽  
Natalija Buza-Vidas ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
Lineage Commitment ◽  
Mouse Bone Marrow ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Stage Of Development ◽  
Number Of Genes

Abstract We recently identified a novel Lin−Sca-1+c-kithiCD34+Flt3hi (LSKCD34+Flt3hi) lymphoid-primed multipotent progenitor (LMPP) in adult mouse bone marrow which, although possessing a combined lymphoid (B and T cell) and myeloid (granulocyte-monocyte; GM) differentiation potential, have little or no ability to adopt erythroid (E) and megakaryocyte (MK) lineage fates (Adolfsson et al, Cell121:295, 2005). The identification of this lineage restricted lymphomyeloid progenitor implicates the existence of alternative roadmaps for lineage commitment of pluripotent hematopoietic stem cells (HSCs), distinct from the classical model suggesting that the first HSC commitment step results in a strict separation into common lymphoid and myeloid progenitors. Herein we provide further, genetic evidence for such a model. Affymetrix global gene profiling, quantitative PCR, and multiplex single cell PCR analysis of LSKCD34−Flt3− long-term (LT)-HSCs, LSKCD34+Flt3− short-term (ST)-HSCs and LSKCD34+Flt3hi LMPPs, demonstrate that LMPPs in contrast to LT-HSCs and ST-HSCs down-regulate or turn off a number of genes critically involved in MkE lineage development, including GATA-1 and the receptors for erythropoietin and thrombopoietin. In contrast, a number of genes specific for early lymphoid development, including Rag-1, sterile Ig and IL-7 receptor are upregulated in LMPPs but absent in LT-HSCs and ST-HSCs. Importantly, within the LMPP, these lymphoid genes are typically co-expressed with a number of GM associated genes such as G-CSF receptor and MPO, but virtually never co-expressed with MkE associated genes. Investigating fetal liver day 14.5 we also provide evidence for existence of the LSKCD34+Flt3hi LMPPs at this early stage of development, and using a single cell clonal assay promoting combined B, T and myeloid lineage development, we demonstrate that a large fraction of fetal LMPPs lacking MkE potential possess a combined GM, B and T cell potential. Thus, evaluation at the single cell level of combined lineage potentials and multilineage gene expression provide compelling evidence for lymphoid-priming within the HSC compartment being preceeded by a loss of MkE potential, but occurring prior to loss of GM potential.

Lymphoid Lineage Restriction in Hematopoietic Stem Cells through Their First Division.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 368-368
Author(s):  
Hideo Ema ◽  
Makoto Kaneda ◽  
Azusa Maeda ◽  
Hina Takano ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Asymmetric Division ◽  
Lineage Commitment ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Lymphoid Lineage ◽  
Lineage Differentiation ◽  
B Lineage

Abstract Little is known of how hematopoietic stem cells (HSCs) differentiate. We have previously suggested that particular myeloid lineages, such as a neutrophil/macrophage (nm) lineage, arise asymmetrically through their first division. In this study, we asked whether lymphoid lineage restriction similarly takes place at the level of HSCs. CD34−c-Kit+Sca-1+lineage−(KSL) cells and CD34+KSL cells, respectively, highly enriched for long-term and short-term repopulating cells, were isolated from bone marrow (BM) of B6 mice by FACS. These cells were subjected to in vitro colony assay and in vivo repopulation assay with use of Rag-2-deficient BM cells, instead of normal BM cells, as competitor cells to facilitate the detection of lymphoid lineage reconstitution. Single CD34−KSL cells were allowed to divide once in the presence of SCF+TPO or SCF+IL-3 under serum-free culture condition. Resultant paired daughter (PD) cells were subsequently separated by micromanipulation and individually transplanted into lethally irradiated mice. Recipient mice were analyzed 7 weeks after transplantation to detect myeloid (My), B-lymphoid (B), and T-lymphoid (T) lineage repopulation by single test donor cells. When single CD34−KSL cells were injected into lethally irradiated mice, about 20% of the cells were detected as MyBT lineage repopulating cells. Interestingly, about 25% of the cells were detected as MyT lineage repopulating cells without detectable level of B lineage reconstitution. Limiting dilution analysis of CD34+KSL cells estimated the frequency of B lineage repopulating cells as close to 1 in 10 cells and that of T lineage repopulating cells as about 1 in 80 cells. Because most colony forming cells among CD34+KSL cells exclusively gave rise to nm lineage, it was assumed that the differentiation potential of CD34+KSL cells was mostly restricted to nm and B lineages. These data suggest more MyT repopulating cells present in CD34− fraction than in CD34+ fraction and, in contrast, more MyB repopulating cells present in CD34+ fraction than in CD34− fraction among KSL cells. On the other hand, My lineage repopulation was observed in about 60% of the PD cells. B and T, B, or T lineage differentiation potential was also detected in approximately 8%, 8%, or 40% of the PD cells with My lineage repopulating activity when generated in the presence of SCF+TPO and in 2%, 5%, or 2% of the cells when generated in the presence of SCF+IL-3. As compared with freshly isolated CD34−KSL cells, T, but not B lineage differentiation potential was well maintained in PD cells by SCF+TPO whereas either B or T lineage differentiation potential was hardly maintained in PD cells by SCF+IL-3. These data indicate the instructive role of cytokines in the restriction of lymphoid potential in HSCs. MyT lineage repopulating cells appeared to be directly generated from MyBT stem cells via their asymmetric division. Taken together, we conclude that asymmetric division of HSCs results in their lymphoid lineage restriction and that T lineage commitment takes place at the level of HSCs, independent of and prior to B lineage commitment which occurs at later stages of differentiation. We propose a novel differentiation model of HSCs which challenges the CLP and CMP based model.

scholarly journals Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3704-3704
Author(s):  
Aldona A Karaczyn ◽  
Edward Jachimowicz ◽  
Jaspreet S Kohli ◽  
Pradeep Sathyanarayana
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Quiescent State ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. Disclosures No relevant conflicts of interest to declare.

Myeloid Lineage Restriction Is Prior to and Independent of Lymphoid Lineage Restriction.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4174-4174
Author(s):  
Azusa Matsubara ◽  
Jun Ooehara ◽  
Yuji Yamazaki ◽  
Hina Takano ◽  
Hiromitsu Nakauchi ◽  
...  
Keyword(s):  
Single Cell ◽  
Lineage Commitment ◽  
Mouse Bone Marrow ◽  
Short Term ◽  
Differentiation Potential ◽  
Myeloid Lineage ◽  
Hematopoietic Stem ◽  
Lymphoid Lineage ◽  
Branched Point

Abstract It has been widely accepted that hematopoietic stem cells (HSCs) exclusively give rise to either myeloid or lymphoid lineage along their differentiation. Phenotypically defined as FcRloCD34+IL-7R−Sca-1−Kit+Lin− cells, common myeloid progenitors (CMPs) are supposedly at the first branched point for myeloid lineage commitment. Phenotypically defined as Thy1.1−IL-7R+KitloSca-1loLin− cells, common lymphoid progenitors are supposedly at the first branched point for both B- and T-lymphoid lineage commitment. Contradicting this model, we previously made observations of the myeloid lineage restriction in very early stages of HSC differentiation. We therefore decided to compare the myeloid and lymphoid differentiation potentials in long-term HSCs, short-term HSCs, multipotent progenitors (MPPs), CMPs, granulocyte/macrophage progenitors (GMPs), megakaryocyte/erythrocyte progenitors (MEPs), and CLPs at the single cell level. Because any available assays are not so sensitive enough for detection of lymphoid lineage differentiation potential, we primarily focused on myeloid differentiation potentials in this study. Here we provide data indicative of the absence of CMPs in the adult mouse bone marrow. Long-term HSCs (CD34−Kit+Sca-1+Lin− cells), short-term HSCs (Flt-3−CD34+Kit+Sca-1+Lin− cells), MPPs (Flt-3+CD34+Kit+Sca-1+Lin− cells), CMPs (FcRloCD34+IL-7R−Sca-1−Kit+Lin− cells), GMPs (FcRhiCD34+IL-7R−Sca-1−Kit+Lin− cells), MEPs (FcRloCD34−IL-7R−Sca-1−Kit+Lin− cells), and CLPs (IL-7R+KitloSca-1loLin− cells) were purified from adult B6 mice by flow cytometry. Single cell cultures were performed in the presence of SCF+TPO+IL-3+EPO. All colonies made by single cells were subjected to Cytospin preparations, followed by May-Gruenwald-Giemsa staining, for morphological classifications of colony cells: neutrophil (n), macrophage (m), erythroblast (E), or megakaryocyte (M). Cells with all n, m, E, M differentiation potentials (nmEM cells) were detected in on average 31%, 5%, and fewer than 1 % of long-term HSCs, short-term HSCs, and MPPs, repectively. Notably, none of a total of over 800 CMPs showed the full nmEM differentiation potential. These CMPs instead showed potentials belonging to members of GMPs and MEPs, suggesting CMPs are mostly an overlaping population of GMPs and MEPs rather than a distinct population. Single cell transplantation experiments revealed the coexistence of cells with the myleoid and B-lymphoid potentials or cells with the myeloid and T-lymphoid potentials among CD34−Kit+Sca-1+Lin− long-term HSC population. These progentiors are likely to be immediate progeny of HSCs. Together, these data support our view that myeloid lineage restriction takes place prior to and independent of lymphoid lineage restriction.

scholarly journals Diabetes impairs the interactions between long-term hematopoietic stem cells and osteopontin-positive cells in the endosteal niche of mouse bone marrow

2013 ◽  
Vol 305 (7) ◽  
pp. C693-C703 ◽  
Author(s):  
Hironori Chiba ◽  
Koji Ataka ◽  
Kousuke Iba ◽  
Kanna Nagaishi ◽  
Toshihiko Yamashita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Mouse Bone Marrow ◽  
Diabetic Mice ◽  
Hematopoietic Stem ◽  
Coculture System ◽  
Endosteal Niche

Hematopoietic stem cells (HSCs) are maintained, and their division/proliferation and quiescence are regulated in the microenvironments, niches, in the bone marrow. Although diabetes is known to induce abnormalities in HSC mobilization and proliferation through chemokine and chemokine receptors, little is known about the interaction between long-term HSCs (LT-HSCs) and osteopontin-positive (OPN) cells in endosteal niche. To examine this interaction, LT-HSCs and OPN cells were isolated from streptozotocin-induced diabetic and nondiabetic mice. In diabetic mice, we observed a reduction in the number of LT-HSCs and OPN cells and impaired expression of Tie2, β-catenin, and N-cadherin on LT-HSCs and β1-integrin, β-catenin, angiopoietin-1, and CXCL12 on OPN cells. In an in vitro coculture system, LT-HSCs isolated from nondiabetic mice exposed to diabetic OPN cells showed abnormal mRNA expression levels of Tie2 and N-cadherin. Conversely, in LT-HSCs derived from diabetic mice exposed to nondiabetic OPN cells, the decreased mRNA expressions of Tie2, β-catenin, and N-cadherin were restored to normal levels. The effects of diabetic or nondiabetic OPN cells on LT-HSCs shown in this coculture system were confirmed by the coinjection of LT-HSCs and OPN cells into bone marrow of irradiated nondiabetic mice. Our results provide new insight into the treatment of diabetes-induced LT-HSC abnormalities and suggest that the replacement of OPN cells may represent a novel treatment strategy.

hFlt3 Expression Marks Long-Term Hematopoietic Stem Cells, but Not the Pathway Common to Granulocyte/Monocyte and Lymphoid Lineages in Human Hematopoiesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1654-1654
Author(s):  
Yoshikane Kikushige ◽  
Goichi Yoshimoto ◽  
Toshihiro Miyamoto ◽  
Fumihiko Ishikawa ◽  
Hiromi Iwasaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Critical Role ◽  
Tyrosine Kinase Receptor ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Low Level ◽  
Flt3 Expression

Abstract Flt3 is a tyrosine kinase receptor, whose signals support proliferation and differentiation of early hematopoietic progenitors in cooperation with other cytokine signals. The concept of myeloid vs. lymphoid commitment in murine hematopoiesis has been challenged based on the analysis of Flt3 expression (Adolfsson et al, Cell, 2006): Long-term murine hematopoietic stem cells (HSCs) with self-renewal activity do not express murine Flt3 (mFlt3), but mFlt3 is upregulated in a fraction of progenitors common to granulocyte/monocyte and lymphoid lineages, suggesting the existence of the 3rd pathway of murine hematolymphoid development. In order to test whether Flt3 expression can delineate such a developmental pathway also in human hematopoiesis, we have analyzed the expression of human Flt3 (hFlt3) in prospectively-purified human stem and progenitors (PNAS 99, 2002) by utilizing 7-color FACS and a highly efficient xenograft systems. Surprisingly, all single human hCD34+hCD38-Thy-1+Lin-long-term HSCs expressed Flt3 at a low level. Purified Flt3lo HSCs can reconstitute NOD-scid/IL2Rγnull newborns (Blood 106, 2005) for a long-term after adoptive transfer of only 100 cells, and generated all hematopoietic cells including GM, megakaryocyte/erythrocyte (MegE), T, B and NK cells, providing formal proof that hFlt3 is expressed in long-term multipotent human HSCs. In the myeloid pathway, hCD34+hCD38+hCD45RA-IL-3RαloLin-CMPs expressed low to negative levels of hFlt3, and hCD34+hCD38+CD45RA-IL-3Rα-Lin-MEPs did not express Flt3. However, in marked contrast to murine progenitors, hCD34+hCD38+hCD45RA+IL-3RαloLin-GMPs uniformly expressed hFlt3 at the highest level. We then separated CMPs into 2 fractions with negative and low level expression of Flt3. In vitro culture in the presence of cytokine cocktail revealed that hFlt3lo CMPs differentiated into hFlt3-CMPs, hFlt3-MEPs and hFlt3hi GMPs, while hFlt3-CMPs mainly generated hFlt3-MEPs in vitro, suggesting that hFlt3lo CMPs is located upstream of the hFlt3-CMP stage that has skewed differentiation potential into the MegE lineage. In vitro differentiation activity of these progenitors was not affected with or without hFlt3 ligand in the culture. hFlt3 ligand, however, appeared to be able to maintain survival of hFlt3-expressing stem and progenitor cells at least in vitro. Thus, in striking contrast to mice, hFlt3 expression is already initiated at the most primitive long-term HSC stage, and is progressively upregulated in accordance with GM lineage differentiation. These data collectively suggest that the role of Flt3 signaling in hematopoietic development might be quite different between human and mouse, and that hFlt3 signaling may especially play a critical role in maintenance of long-term HSCs and in development of GM cells in human hematopoiesis.

scholarly journals Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Elvin Wagenblast ◽  
Maria Azkanaz ◽  
Sabrina A. Smith ◽  
Lorien Shakib ◽  
Jessica L. McLeod ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Single Cell ◽  
High Efficiency ◽  
Hematopoietic Stem ◽  
Functional Profiling ◽  
Differentiation And Proliferation ◽  
Gata1 Transcription Factor

Abstract In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.

Identification of a Novel Murine CD45 Negative Bone Marrow-Derived Cell Population with In Vivo Marrow Repopulating Potential Following Hematopoietic Specification In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1719-1719
Author(s):  
Edward F. Srour ◽  
Tamara L. Horvath
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Pcr Analysis ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow

Abstract Murine bone marrow-derived cells expressing Sca-1+c-kit+lin− (KSL), as well as subfractions of these cells, represent an enriched population of hematopoietic stem cells (HSC) capable of long-term reconstitution of lethally irradiated recipients. Commitment to the hematopoietic lineage is invariably associated with expression of the pan-leukocyte marker CD45 which is also expressed on KSL cells. Whether KSL cells are the most primitive population of HSC present in the bone marrow (BM) is not fully resolved. We hypothesized that putative HSC that are more primitive than KSL cells may not express CD45 or genetic elements that mark early hematopoietic specification and commitment, but may mature under appropriate conditions into CD45+ cells capable of hematopoietic differentiation in conditioned hosts. BM cells from 8 to 10-week old BoyJ mice were collected by flushing and erythrocytes were lysed. The remaining cells were stained and sorted to yield CD45+ Sca-1+ c-kit+ (CD45+HSC) and CD45− Sca-1+ c-kit− (CD45−) cells which represented approximately 0.02% of total cells analyzed. PCR analysis of both cell populations revealed that CD45+HSC expressed CD45 and SCL but not PU.1 while CD45− cells did not express any of these genes. Directly after sorting, CD45+HSC, but not CD45− cells contained clonogenic cells that gave rise to hematopoietic colonies in progenitor cell assays. Similarly, while fresh CD45+HSC were able to respond to exogenous hematopoietic cytokines including SCF, TPO, and FL in liquid suspension cultures as evidenced by expansion and differentiation, their CD45− counterparts failed to proliferate under these conditions and none survived beyond 7 days of culture. When transplanted competitively into lethally irradiated congenic recipients, only freshly isolated CD45+HSC sustained donor-derived hematopoiesis, whereas hematopoiesis in mice injected with freshly isolated CD45− cells was sustained long term by competitor cells and endogenous host-derived stem cells. Both groups of CD45+HSC and CD45− cells could be expanded on irradiated M210B4 stromal cells when supplemented with SCF, TPO, and FL, with CD45− cells giving rise to cobblestone foci of small, round translucent cells beginning on day 7 of culture. Cultured CD45+HSC continued to express CD45 and SCL and, depending on the length of culture, also expressed PU.1. Interestingly, after 15 days in culture, CD45− cells expressed CD45 by RT-PCR and FACS (in addition to Sca-1) and also expressed mRNA for SCL. Given the ability of CD45− cells to expand under these conditions and to acquire CD45 expression, we next compared the repopulating potential of fresh and cultured CD45+HSC and CD45− cells using lethally irradiated C57Bl/6 recipients. As expected, fresh CD45+HSC sustained donor-derived engraftment and culture of these cells over M210B4 for 15 days reduced their repopulating potential more than 7-fold. In contrast, CD45− cells maintained on M210B4 (the expansion equivalent of 750 cells seeded) contributed to hematopoietic engraftment, albeit at low levels (under 5% chimerism). These data demonstrate that CD45− Sca-1+ c-kit− cells may be marrow resident precursors of hematopoietic stem cells and suggest that early stages of the HSC hierarchy may include CD45− cells. Whether these CD45− cells also posses endothelial differentiation potential and can give rise to CD45+HSC in vivo is now under investigation.

Unexpected and Sustained Symmetry in the Repopulation Programs Displayed by Clonally Amplified Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1651-1651
Author(s):  
Brad Dykstra ◽  
David Kent ◽  
Melisa Hamilton ◽  
Merete Kristiansen ◽  
Kristin Lyons ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Adult Life ◽  
Mouse Bone Marrow ◽  
Recipient Mouse ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Over Time

Abstract Heterogeneity in progeny output by individual pluripotent hematopoietic cells is a well documented but poorly understood paradigm. Importantly, the extent to which this functional heterogeneity is pre-determined by intrinsic mechanisms that specify distinct programs, as opposed to conditions that result in a series of stochastic events, is still debated. The prospective isolation of phenotypically defined subpopulations with more restricted behaviors has lent recent support to the concept of predetermined hierarchies with preset, but alternative pathways of lineage restriction and differentiated cell output. Here we have used highly purified starting populations to compare the long-term cell output dynamics of individual multipotent repopulating cells in sublethally irradiated W41/W41 mice transplanted with single Ly-5 congenic CD45midlin−Rho−SP adult mouse bone marrow cells (158 mice) or their clonal progeny generated after 4 days in vitro in 300 ng/ml SF, 20 ng/ml IL-11 and 1 ng/ml Flt3-L (194 mice). WBC samples collected 4, 8, 12, 16, and 24 weeks post-transplant were analyzed for donor contributions to the myeloid (GM) and lymphoid (B and T) lineages. In 49 of the 158 mice (31%) and 44 of the 194 mice (23%), the cells produced in vivo contributed ≥1% of all the WBCs present at ≥16 weeks. The overall and lineage-specific contributions to the WBCs in each recipient mouse varied widely both over time post-transplant and between mice. However, examination of the ratio of the donor contributions to the myeloid and lymphoid lineages (GM:B+T) in each mouse at 16 weeks post-transplant allowed 4 patterns to be readily identified: α and β with GM:B+T ratios of ≥2 and 0.25–2, respectively; γ, with a GM:B+T ratio of <0.25 including a ≥1% contribution to both lymphoid and myeloid lineages at 16 weeks; and δ, also with a GM:B+T ratio of <0.25, but with contribution only to the lymphoid lineages at this time. Secondary transplants performed after 24 weeks showed long-term repopulation (≥16 weeks) of most recipients of type α and β progeny (10/11 and 11/12, respectively) but none of the recipients of type γ and δ progeny were repopulated (0/6 and 0/17, respectively). Interestingly, the variation over time in both the overall and lineage-specific contributions was remarkably similar in pairs of secondary recipients injected with cells from the same primary donor. In addition, the lineage contribution ratios seen in the secondary recipients tended to recapitulate that of the primary donors (i.e., α or β), and these trends remained obvious when tertiary transplants were performed. Preservation of stem cell programming was also evident from sequential analyses of multiple mice injected with aliquots of the same clones generated in vitro after 10 days from single CD45midlin−Rho−SP cells. Very similar patterns of total and lineage-specific contributions were again observed amongst the different recipients of cells from the same clones. Collectively, these findings indicate that by early adult life hematopoietic stem cells have acquired intrinsically fixed patterns of lineage specification that can be stably transmitted through many self-renewal generations.

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