Development of Multi-Lineage Hematopoiesis in Two Novel Zebrafish runx1 mutants

Abstract Long term hematopoietic stem cells are essential for the life-long maintenance of the hematopoietic system of an organism. The transcription factor RUNX1 is required for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium during the embryo development. Runx1 knockout mouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. However, we previously showed that zebrafish homozygous for an ENU-induced nonsense mutation in runx1 (runx1W84X/W84X) were able to recover from a larval "bloodless" phase and develop to fertile adults with multi-lineage hematopoiesis, suggesting the formation of runx1-independent adult HSCs. However, our finding was based on a single zebrafish model, which requires verification in additional, independent models. In order to further investigate if a RUNX1-independent pathway exists for the formation of adult HSCs, we generated two new runx1 mutants, a deletion of 8 bp (runx1del8/del8) and a deletion of 25 bp (runx1del25/del25) within exon 4 of runx1, respectively, using the Transcription activator-like effector nucleases (TALENs) technology. These mutations cause frameshifts and premature terminations within the runt-homology domain,, resulting in loss of function of runx1 (runx1-/-). Both runx1del8/del8 and runx1del25/del25 mutant embryos had normal primitive hematopoiesis but failed to develop definitive hematopoiesis. Time-lapse recordings with confocal microscopy revealed that, indeed, there was no emergence of HSCs from the ventral wall of dorsal aorta in the runx1-/- embryos. The runx1-/- larvae gradually lost circulating primitive blood cells and became bloodless between 8 and 14 days post fertilization (dpf). However they gradually regained circulating blood cells between 15 and 20 dpf. Eventually, about 40% of runx1del8/del8 and runx1del25/del25 mutants developed to fertile adults with circulating blood cells of multi-lineages. Taken together, our data is consistent with the previously described runx1W84X/W84X phenotype and supports the possibility of a runx1-independent mechanism for HSC formation and definitive hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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Deletion of Hoxa Genes in Adult Mice Affects Hematopoietic Stem Cell and B Cell Progenitors

Blood ◽

10.1182/blood.v118.21.2359.2359 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2359-2359

Author(s):

Charles-Etienne Lebert-Ghali ◽

Marilaine Fournier ◽

Heloise Frison ◽

Janetta Jacoba Bijl

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

B Cell ◽

Hox Genes ◽

Conflicts Of Interest ◽

Proliferative Potential ◽

Hoxa Cluster ◽

Hematopoietic Stem ◽

Hoxa Genes ◽

Definitive Hematopoiesis

Abstract Abstract 2359 BACKGROUND AND OBJECTIVE: Functional compensation between homeodomain proteins has hindered the ability to unravel their role in hematopoiesis using single gene knock-outs. Although several Hox genes can expand hematopoietic stem cells (HSC) when overexpressed, it remains unclear whether these genes are required for proper adult hematopoiesis. Moreover, it has been shown that HoxB genes are dispensable for hematopoiesis, and that expression of most HoxA genes is ten-fold superior to genes from other Hox clusters in HSC enriched fetal liver populations (Bijl, 2006). Using a haploinsufficient mice for the entire HoxA cluster (HoxA+/−), we have shown that adult HSCs and progenitors are particularly sensitive to HoxA gene levels (Lebert-Ghali, 2010). Thus, we hypothesize that HoxA genes have a crucial function in definitive hematopoiesis. MATERIALS AND METHODS: To assess the role of HoxA genes in definitive hematopoiesis, we used a conditional mutant mouse model for the entire HoxA cluster in combination with an inducible Mx-Cre model. The functional effect of HoxA cluster deletion on hematopoietic cells was analysed by culture and repopulation assays. RESULTS: Highly efficient excision of HoxA cluster was achieved by 7 doses of poly(I):poly(C) treatment (91–100%). Mice (control, n=3 and Mx-CreHoxAflox/flox, n=3) were sacrificed and analysed three days after the last injection. Immunophenotyping showed a 3 to 4 fold increase of CD150+/CD48-/CD244-/Sca+/c-kit+/Lin- hematopoietic stem cells. Despite the enhancement of the HoxA−/− HSC population, single cell cultures showed that their proliferative potential in response to growth factors was significantly reduced (p=0.036) as growth was observed only for 16.6 ± 14.4% of HoxA−/− compared to 42.4 ±10.3% of control HSCs after 3-weeks of culturing. In contrast, the number of multipotent progenitor (MPP) cells (CD34+/CD135+/Sca+/c-kit+/Lin-) was reduced, indicating a partial block from the short-term HSC (CD34+/CD135-/Sca+/c-kit+/Lin-) to the MPP transition. Colony forming cell assays showed a dramatic decrease of B-cell progenitors in the bone marrow (BM) (10-fold, p=0.0079), while myeloid progenitors were not affected by the deletion. Transplantation assays demonstrated that grafts composed of > 91% HoxA−/− HSCs have slower repopulation kinetics compared to control HSCs and strongly reduced long-term engraftment (37 ± 28% and 92 ± 6% for HoxA−/− and control, respectively, 20 weeks post-transplantation). Genotyping of engrafted donor cells is currently analyzed to confirm repopulation by HoxA−/− cells. Consistent with the observations in primary mice, peripheral blood analysis revealed also a dramatic reduction of B220+ B-cell population in mice transplanted with HoxA−/− BM cells compared to control (7.1 ± 8.5% and 56.2 ±4.8% respectively p=0,000002) Altogether, in vitro assays and transplantation assays revealed that the functions of HoxA−/− HSC seem to be impaired. CONCLUSION: Together, these results show that HoxA cluster genes are required for both HSC function and B cell development, indicating that these genes are important regulators of adult hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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MSC-Derived HSCs Can Reconstitute Hematopoietic Function In Patients With Severe Aplastic Anemia

Blood ◽

10.1182/blood.v122.21.2432.2432 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2432-2432 ◽

Cited By ~ 1

Author(s):

James Q Yin ◽

Chunji Gao ◽

Bing Han ◽

Jianliang Sheng

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Hematopoietic Stem Cells ◽

Aplastic Anemia ◽

Blood Cells ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Severe Aplastic Anemia ◽

Hematopoietic Stem

Abstract Introduction Naturally-occurring regeneration of cells and tissues is generally involved in four working mechanisms such as directed differentiation, dedifferentiation, trans-differentiation and transdetermination. The better exploring of these mechanisms could be beneficial to develop clinical strategies for regenerative medicine and to reduce the likelihood of immune rejection and relevant complications Recently, “trans-determination” has attracted great controversy, mostly in regards to whether adult stem cells can colonize other tissues after transplantation. More importantly, how to generate large amounts of a particular stem cell type through a transdetermination process remained to be unsolved. Similarly, it is unclear whether mesenchymal stem cells (MSCs) can transdeterminate into hematopoietic stem cells (HSCs). Methods Many technologies were used to validate the transdetermination of adipose-derived mesenchymal stem cells (AD-MSCs) into hematopoietic stem cells (HSCs) from different aspects. They include FACS analysis, PCR tests, immunostaining, expansion and repopulating assays, transplantation analysis and others, showing their in vivo and in vitro potentials for long-term self-renewal and differentiation into multi-lineages of blood cells. Moreover, these AD-HSCs can reconstitute hematopoietic function in six patients. Results We report firstly here that a huge number of human AD-MSCs that are CD44+,CD29+, CD105+, CD166+,CD133-,CD34- could rapidly transdifferentiate into hematopoietic stem cells (CD49f+/CD133+/CD34+) and their descending blood cells in vitro, after transfected with two small RNAs. The sRNAs were high-effectively delivered into MSCs by a novel peptide means. These adipose-derived HSCs (AD-HSCs) could form different types of hematopoietic colonies as nature-occurring HSCs did. Upon the primary and secondary transplantation into sublethally or lethally irradiated mice, these MSC-HSCs engrafted and differentiated into all hematopoietic lineages such as erythrocytes, lymphocytes, myelocytes and thrombocytes. Furthermore, we demonstrated the first evidence that the transdetermination of MSCs was induced by acetylation of histone proteins and activation of many transcriptional factors. More excitingly, these MSC-derived HSCs can reconstitute hematopoietic function in six patients with severe aplastic anemia. Conclusion our findings identify the molecular mechanisms that regulate the directed transdifferentiation of MSCs toward HSCs, create a new source for individual HSC transplantation used for the treatment of blood diseases and cancers, and break the stalemate caused by bone marrow match and graft-versus-host disease. Disclosures: No relevant conflicts of interest to declare.

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gata2is Required for the runx1-Independent Hematopoiesis in Zebrafish

Blood ◽

10.1182/blood-2019-127334 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2463-2463

Author(s):

Erica Bresciani ◽

Blake Carrington ◽

Erika M Kwon Kim ◽

Kai Yu ◽

Kevin Bishop ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Hematopoietic Stem Cells ◽

Compensatory Mechanism ◽

Loss Of Function ◽

Wild Type ◽

Hematopoietic Stem ◽

Larval Stages ◽

Independent Mechanism ◽

Definitive Hematopoiesis

The current notion about how hematopoietic stem cells (HSCs) are generated identifies the transcription factor RUNX1 as an essential factor for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium. Consequently, Runx1knockout mice fail to develop definitive hematopoiesis and lack all definitive blood lineages and cannot survive past embryonic day 12. However, even though zebrafish with arunx1stop codon mutation (runx1W84X/W84X) presented defects in definitive hematopoiesis during embryogenesis, runx1W84X/W84Xembryos could develop to fertile adults with blood cells of multi-lineages, raising the possibility that HSCs can emerge without RUNX1. In order to determine if a RUNX1-independent mechanism can support the generation of HSCs we have generated three new zebrafish runx1-/- with engineered deletions of the runx1gene using TALEN and CRISPR-Cas9. Our analysis shows that all three mutants have identical phenotypei.e., failure to develop definitive hematopoiesis during early embryogenesis, with later reemergence of hematopoietic cells and survivalof therunx1 mutants to adulthood, further confirming the existence of a RUNX1-independent mechanism for the emergence of HSCs. In the absence of a functional runx1, a cd41-GFP+population of hematopoietic precursors can still be detected in the aorta-gonad-mesonephros (AGM) region and in the hematopoietic tissues of the mutant embryos. Single cell RNA sequencing of the wild type and mutant HSC/HSPC at embryonic and larval stages confirmed the presence of a population of runx1- /-cd41:GFPlow cells expressing HSC signature genes at 2.5 days post fertilization. At larval stages the runx1-/-HSCs maintain their ability to generate erythroid and myeloid lineage progenitors but they present a different expression profile compared to the wild type. In order to uncover the compensatory mechanism that drives the repopulation of the hematopoietic compartment in the absence of runx1we identified the molecular signatures that separate the runx1-/-HSC/HSPCs from the wild type and subsequently focused our attention on the transcription factors differentially expressed in the runx1-/-HSC/HSPCs. Our analysis shows that the master transcription factor gata2b is strongly upregulated in the runx1- /-HSCs during the recovery of hematopoiesis and it is also upregulated in the kidney marrow of the surviving runx1-/-adults. Given the key role of GATA2 in the HSC development and maintenance in both mouse and zebrafish, gata2b represented a strong candidate gene with the potential ability to drive the rescue of the runx1-/-phenotype. Indeed, a loss of function mutation or knock-down of gata2b can significantly reduce or abolish the survivability of the runx1-/-fish, indicating that gata2bis responsible for rescuing hematopoiesis in the runx1 mutant fish. Overall our results show that even though runx1 is necessary for the normal emergence of definitive HSCs in the embryos, in the absence of runx1the transcription factor gata2 is able to support definitive hematopoiesis that is sufficient for the embryos to develop to functional adults in the zebrafish. The current notion about how hematopoietic stem cells (HSCs) are generated identifies the transcription factor RUNX1 as an essential factor for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium. Consequently, Runx1knockout mice fail to develop definitive hematopoiesis and lack all definitive blood lineages and cannot survive past embryonic day 12. However, even though zebrafish with arunx1stop codon mutation (runx1W84X/W84X) presented defects in definitive hematopoiesis during embryogenesis, runx1W84X/W84Xembryos could develop to fertile adults with blood cells of multi-lineages, raising the possibility that HSCs can emerge without RUNX1. In order to determine if a RUNX1-independent mechanism can support the generation of HSCs we have generated three new zebrafish runx1-/- with engineered deletions of the runx1gene using TALEN and CRISPR-Cas9. Our analysis shows that all three mutants have identical phenotypei.e., failure to develop definitive hematopoiesis during early embryogenesis, with later reemergence of hematopoietic cells and survivalof therunx1 mutants to adulthood, further confirming the existence of a RUNX1-independent mechanism for the emergence of HSCs. In the absence of a functional runx1, a cd41-GFP+population of hematopoietic precursors can still be detected in the aorta-gonad-mesonephros (AGM) region and in the hematopoietic tissues of the mutant embryos. Single cell RNA sequencing of the wild type and mutant HSC/HSPC at embryonic and larval stages confirmed the presence of a population of runx1- /-cd41:GFPlow cells expressing HSC signature genes at 2.5 days post fertilization. At larval stages the runx1-/-HSCs maintain their ability to generate erythroid and myeloid lineage progenitors but they present a different expression profile compared to the wild type. In order to uncover the compensatory mechanism that drives the repopulation of the hematopoietic compartment in the absence of runx1we identified the molecular signatures that separate the runx1-/-HSC/HSPCs from the wild type and subsequently focused our attention on the transcription factors differentially expressed in the runx1-/-HSC/HSPCs. Our analysis shows that the master transcription factor gata2b is strongly upregulated in the runx1- /-HSCs during the recovery of hematopoiesis and it is also upregulated in the kidney marrow of the surviving runx1-/-adults. Given the key role of GATA2 in the HSC development and maintenance in both mouse and zebrafish, gata2b represented a strong candidate gene with the potential ability to drive the rescue of the runx1-/-phenotype. Indeed, a loss of function mutation or knock-down of gata2b can significantly reduce or abolish the survivability of the runx1-/-fish, indicating that gata2bis responsible for rescuing hematopoiesis in the runx1 mutant fish. Overall our results show that even though runx1 is necessary for the normal emergence of definitive HSCs in the embryos, in the absence of runx1the transcription factor gata2 is able to support definitive hematopoiesis that is sufficient for the embryos to develop to functional adults in the zebrafish. Disclosures No relevant conflicts of interest to declare.

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Platelet Depletion Causes Rapid But Transient Activation Of Mouse Bone Marrow Long-Term Hematopoietic Stem Cells To Participate In Stress Thrombopoiesis

Blood ◽

10.1182/blood.v122.21.2436.2436 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2436-2436

Author(s):

Lijian Shao ◽

Di Hou ◽

Wei Feng ◽

Jianhui Chang ◽

Jerry Ware ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Blood Platelet ◽

Small Population ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Platelet Depletion

Abstract Hematopoietic stem cells (HSCs) are responsible for the production of various lineages of blood cells throughout life. To ensure the longevity of HSCs and prevent their premature exhaustion, a small population of HSCs termed long-term HSCs (LT-HSCs) is maintained in quiescence under homeostatic conditions. Whether LT-HSCs can exit quiescence rapidly in response to hematopoietic stress to replenish blood cells is not known, and thus was investigated in the present study in an acute but transient platelet depletion mouse model induced by intravenous injection of anti-GPIbα antibody. The results from our study show that in response to platelet depletion LT-HSCs can exit quiescence promptly and then proliferate rapidly to participate in stress thrombopoiesis probably via an alternative differentiation pathway. The mechanism by which platelet depletion causes LT-HSC activation is not due to a direct effect of the antibody on LT-HSCs or activation of platelets, but is attributed to a transient reduction of thrombopoietin (TPO) resulting from the acute depletion of platelets. However, the activated LT-HSCs return to quiescence when blood platelet counts are almost back to normal levels without compromising their function. These findings suggest that there is a very efficient and sensitive feedback regulatory circuitry between quiescent LT-HSCs and platelets, which allows LT-HSCs to directly and promptly respond to hematopoietic stress resulting from an acute loss of platelets. In this response, TPO may function as a switch that can rapidly turn on LT-HSCs to participate in stress thrombopoiesis. Disclosures: No relevant conflicts of interest to declare.

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Ing4-Deficiency Enhances Hematopoietic Stem Cell Quiescence and Confers Resistance to Inflammatory Stress

Blood ◽

10.1182/blood-2021-154353 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1095-1095

Author(s):

Zanshé Thompson ◽

Georgina A Anderson ◽

Seth Gabriel ◽

Melanie Rodriguez ◽

Vera Binder ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Target Genes ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Critical Role ◽

Loss Of Function ◽

Wild Type ◽

Hematopoietic Stem

Abstract In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, loss of Ing4 has been shown to promote stem cell-like characteristics in malignant cells and it is a frequent target of inactivation in various types of cancer. Mutations in Ing4 cause deregulation of both NF-kB and c-Myc target gene expression. We have also identified a requirement for Ing4 in murine hematopoiesis. Ing4-/- mice have aberrant hematopoiesis and elevated cytokine expression in bone marrow cells. Using RNA-sequencing, we found that Ing4-deficient HSPCs express high levels of c-Myc target genes and genes associated with oxidative phosphorylation and ribosomal biogenesis. Yet, Ing4 deficiency induces G 0 arrest in HSPCs and they have low levels of reactive oxygen species. This places Ing4-deficient HSPCs in a poised state, where they are quiescent, but express elevated levels of genes associated with differentiation. Under stress hematopoiesis following low-dose irradiation, Ing4-deficient long-term hematopoietic stem cells (LT-HSCs) do not expand, but short-term hematopoietic stem cells (ST-HSCs) function comparably to wild-type. Similarly, under transplantation stress, LT-HSCs fail to contribute to multilineage chimerism, while ST-HSCs contribute at levels equal to wild-type cells. These results are striking, particularly when compared to other models of enhanced NF-kB activity, where HSPCs cannot contribute to multilineage chimerism in transplantation. We sought to target the misregulated pathways in Ing4-deficient HSCs to rescue to effects of Ing4 deficiency. To this end, we chose to target the c-Myc pathway for several reasons: c-Myc target genes are over-represented in our RNA-seq data, c-Myc lies upstream of several of the misregulated pathways observed in Ing4-/- HSCs, and Ing4 has previously been reported to negatively regulate c-Myc activity directly. When treated with the c-Myc inhibitor, 10058-F4, both LT-HSCs and ST-HSCs are pushed into cycling, but this treatment also resulted in fewer cells overall. These results suggest that dampening of the c-Myc pathway can partially rescue Ing4 loss of function. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. Disclosures No relevant conflicts of interest to declare.

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Merocyanine 540-sensitized photoinactivation of human erythrocytes parasitized by Plasmodium falciparum

Blood ◽

10.1182/blood.v80.1.21.bloodjournal80121 ◽

1992 ◽

Vol 80 (1) ◽

pp. 21-24 ◽

Cited By ~ 1

Author(s):

OM Smith ◽

SA Dolan ◽

JA Dvorak ◽

TE Wellems ◽

F Sieber

Keyword(s):

Stem Cells ◽

Plasmodium Falciparum ◽

Hematopoietic Stem Cells ◽

Red Blood Cells ◽

Human Blood ◽

Blood Cells ◽

Potential Usefulness ◽

Hematopoietic Stem ◽

Infected Blood ◽

Merocyanine 540

The purpose of this study was to evaluate the photosensitizing dye merocyanine 540 (MC540) as a means for extracorporeal purging of Plasmodium falciparum-infected erythrocytes from human blood. Parasitized red blood cells bound more dye than nonparasitized cells, and exposure to MC540 and light under conditions that are relatively well tolerated by normal erythrocytes and normal pluripotent hematopoietic stem cells reduced the concentration of parasitized cells by as much as 1,000-fold. Cells parasitized by the chloroquine- sensitive HB3 clone and the chloroquine-resistant Dd2 clone of P falciparum were equally susceptible to MC540-sensitized photolysis. These data suggest the potential usefulness of MC540 in the purging of P falciparum-infected blood.

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Scl isoforms act downstream of etsrp to specify angioblasts and definitive hematopoietic stem cells

Blood ◽

10.1182/blood-2009-09-244640 ◽

2010 ◽

Vol 115 (26) ◽

pp. 5338-5346 ◽

Cited By ~ 42

Author(s):

Xi Ren ◽

Gustavo A. Gomez ◽

Bo Zhang ◽

Shuo Lin

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Critical Role ◽

Vascular Endothelial ◽

Hematopoietic Stem ◽

Vegf Signaling ◽

Endothelial Growth Factor ◽

Endothelial Development ◽

Definitive Hematopoiesis ◽

Runx1 Expression

Abstract Recent lineage studies suggest that hematopoietic stem cells (HSCs) may be derived from endothelial cells. However, the genetic hierarchy governing the emergence of HSCs remains elusive. We report here that zebrafish ets1-related protein (etsrp), which is essential for vascular endothelial development, also plays a critical role in the initiation of definitive hematopoiesis by controlling the expression of 2 stem cell leukemia (scl) isoforms (scl-α and scl-β) in angioblasts. In etsrp morphants, which are deficient in endothelial and HSC development, scl-α alone partially rescues angioblast specification, arterial-venous differentiation, and the expression of HSC markers, runx1 and c-myb, whereas scl-β requires angioblast rescue by fli1a to restore runx1 expression. Interestingly, when vascular endothelial growth factor (Vegf) signaling is inhibited, HSC marker expression can still be restored by scl-α in etsrp morphants, whereas the rescue of arterial ephrinb2a expression is blocked. Furthermore, both scl isoforms partially rescue runx1 but not ephrinb2a expression in embryos deficient in Vegf signaling. Our data suggest that downstream of etsrp, scl-α and fli1a specify the angioblasts, whereas scl-β further initiates HSC specification from this angioblast population, and that Vegf signaling acts upstream of scl-β during definitive hematopoiesis.

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The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells

Nature Immunology ◽

10.1038/ni.1617 ◽

2008 ◽

Vol 9 (7) ◽

pp. 810-819 ◽

Cited By ~ 166

Author(s):

Stephen J Loughran ◽

Elizabeth A Kruse ◽

Douglas F Hacking ◽

Carolyn A de Graaf ◽

Craig D Hyland ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem ◽

Definitive Hematopoiesis

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Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ hematopoietic stem cells

Blood ◽

10.1182/blood.v83.12.3758.3758 ◽

1994 ◽

Vol 83 (12) ◽

pp. 3758-3779 ◽

Cited By ~ 123

Author(s):

N Uchida ◽

HL Aguila ◽

WH Fleming ◽

L Jerabek ◽

IL Weissman

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Blood Cells ◽

Critical Role ◽

White Blood Cells ◽

Cell Types ◽

Dose Dependence ◽

Hematopoietic Stem ◽

Hematopoietic Recovery

Abstract Hematopoietic stem cells (HSCs) are believed to play a critical role in the sustained repopulation of all blood cells after bone marrow transplantation (BMT). However, understanding the role of HSCs versus other hematopoietic cells in the quantitative reconstitution of various blood cell types has awaited methods to isolate HSCs. A candidate population of mouse HSCs, Thy-1.1lo Lin-Sca-1+ cells, was isolated several years ago and, recently, this population has been shown to be the only population of BM cells that contains HSCs in C57BL/Ka-Thy-1.1 mice. As few as 100 of these cells can radioprotect 95% to 100% of irradiated mice, resulting long-term multilineage reconstitution. In this study, we examined the reconstitution potential of irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ BM cells. Donor-derived peripheral blood (PB) white blood cells were detected as early as day 9 or 10 when 100 to 1,000 Thy-1.1lo Lin-Sca-1+ cells were used, with minor dose-dependent differences. The reappearance of platelets by day 14 and thereafter was also seen at all HSC doses (100 to 1,000 cells), with a slight dose-dependence. All studied HSC doses also allowed RBC levels to recover, although at the 100 cell dose a delay in hematocrit recovery was observed at day 14. When irradiated mice were transplanted with 500 Thy-1.1lo Lin-Sca-1+ cells compared with 1 x 10(6) BM cells (the equivalent amount of cells that contain 500 Thy-1.1lo Lin-Sca-1+ cells as well as progenitor and mature cells), very little difference in the kinetics of recovery of PB, white blood cells, platelets, and hematocrit was observed. Surprisingly, even when 200 Thy1.1lo Lin-Sca- 1+ cells were mixed with 4 x 10(5) Sca-1- BM cells in a competitive repopulation assay, most of the early (days 11 and 14) PB myeloid cells were derived from the HSC genotype, indicating the superiority of the Thy-1.1lo Lin-Sca-1+ cells over Sca-1- cells even in the early phases of myeloid reconstitution. Within the Thy-1.1lo Lin-Sca-1+ population, the Rhodamine 123 (Rh123)hi subset dominates in PB myeloid reconstitution at 10 to 14 days, only to be overtaken by the Rh123lo subset at 3 weeks and thereafter. These findings indicate that HSCs can account for the early phase of hematopoietic recovery, as well as sustained hematopoiesis, and raise questions about the role of non-HSC BM populations in the setting of BMT.

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Mesenchymal Stem Cells from the Wharton’s Jelly of Umbilical Cord Segments Support the Maintenance of Long Term Culture-Initiating Cells from Cord Blood.

Blood ◽

10.1182/blood.v108.11.3650.3650 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3650-3650

Author(s):

Kent W. Christopherson ◽

Tiki Bakhshi ◽

Shamanique Bodie ◽

Shannon Kidd ◽

Ryan Zabriskie ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Blood Cells ◽

Hematopoietic Stem ◽

Cord Blood Cells

Abstract Hematopoietic Stem Cells (HSC) are routinely obtained from bone marrow, mobilized peripheral blood, and umbilical Cord Blood. Traditionally, adult bone marrow has been utilized as a source of Mesenchymal Stem Cells (MSC). Bone marrow derived MSC (BM-MSC) have previously been shown to maintain the growth of HSC obtained from cord blood and have been utilized for cord blood expansion purposes. However, the use of a mismatched BM-MSC feeder stromal layer to support the long term culture of cord blood HSC is not ideal for transplant purposes. The isolation of MSC from a novel source, the Wharton’s Jelly of Umbilical Cord segments, was recently reported (Romanov Y, et al. Stem Cells.2003; 21: 105–110) (Lee O, et al. Blood.2004; 103: 1669–1675). We therefore hypothesized that Umbilical Cord derived MSC (UC-MSC) have the ability to support the long term growth of cord blood derived HSC similar to that previously reported for BM-MSC. To test this hypothesis, MSC were isolated from the Wharton’s Jelly of Umbilical Cord segments and defined morphologically and by cell surface markers. UC-MSC were then tested for their ability to support the growth of pooled CD34+ cord blood cells in long term culture - initiating cell (LTC-IC) assays as compared to BM-MSC. We observed that like BM-MSC, CB-MSC express a defined set of cell surface markers. By flow cytometry we determined that that both UC-MSC and BM-MSC are positive for CD29, CD44, CD73, CD90, CD105, CD166, HLA-A and negative for CD45, CD34, CD38, CD117, HLA-DR expression. Utilizing Mitomycin C treated (200 μM, 15 min.) UC-MSC from multiple donors as a feeder layer we observed that UC-MSC have the ability to support the maintenance of long term hematopoiesis during the LTC-IC assay. Specifically, UC-MSC isolated from separate umbilical cord donors support the growth of 69.6±11.9 (1A), 31.7±3.9 (2B), 67.0±13.5 (3A), and 38.5±13.7 (3B) colony forming cells (CFC) per 1×104 CD34+ cord blood cells as compared to 64.0±4.2 CFC per 1×104 CD34+ cord blood cells supported by BM-MSC (Mean±SEM, N=4 separate segments from three different donors). Thus, Umbilical Cord derived Mesenchymal Stem Cells, a recently described novel source of MSC, have the ability to support long term maintenance of Hematopoietic Stem Cells, as defined by the LTC-IC assay. These results may have potential therapeutic application with respect to ex vivo stem cell expansion of Cord Blood Hematopoietic Stem Cells utilizing a Mesenchymal Stem Cell stromal layer. In addition, these data suggest the possibility of co-transplantation of matched Mesenchymal and Hematopoietic Stem Cells from the same umbilical cord and cord blood donor respectively. Lastly, these results describe a novel model system for the future study of the interaction between Cord Blood Hematopoietic Stem Cells and the appropriate supportive microenvironment represented by the Umbilical Cord - Mesenchymal Stem Cells.

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