scholarly journals Long-Term Hematopoietic Clonal Stability Tracked Using Molecular Barcoding in Non-Human Primates

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2908-2908
Author(s):  
Samson Koelle ◽  
Chuanfeng Wu ◽  
Brian Li ◽  
Rong Lu ◽  
Robert E. Donahue ◽  
...  
Keyword(s):  
Stem Cell ◽  
B Cell ◽  
Cell Fate ◽  
High Throughput Sequencing ◽  
Conflicts Of Interest ◽  
Rhesus Macaques ◽  
Small Subset ◽  
Hematopoietic Stem ◽  
Over Time

Abstract By transplanting rhesus macaques with autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) labeled with lentivirally delivered high diversity oligonucleotide barcodes, we are able to track complex clonal contributions to hematopoiesis over time and across lineages via low cycle PCR and high-throughput sequencing of the inserted barcode. We reported on patterns of reconstitution for the first 9 months following engraftment in a recent publication (Wu et al, Cell Stem Cell, 2014). For the first 1 to 3 months short lived unilineage progenitors predominated, followed by increased clonal diversity and the emergence of first shared myeloid (My)/B clones, and then My/B/T clones. A major novel finding was a distinct ontogeny for NK cells, with the major CD16+ blood NK population showing non-overlapping origin with My/B and T lineages through 6-9 months. We now report on hematopoietic clonal patterns up to 23 months post-transplant in four animals. In all four animals, a group of high contributing NK clones contributed minimally to other lineages until 9 to 12 months, at which point these unilineage NK clones extinguished and a new generation of NK high contributors appeared. In two animals, these emerging clones continued to be either unilineage or highly biased towards the NK lineage, while in two other animals, they began to contribute highly to all lineages, a discrepancy which possibly derives from differences between animals in barcode marking level. Over the 17 and 23 months since transplant tracked in the two highly marked animals, 5.6±1.4 and 9±0.9 out of the top 10 NK contributing clones, respectively, were highly biased towards NK and away from other lineages, as determined by unsupervised hierarchical clustering of correlations between high contributing clones. Though shared My/B/T progenitors emerged after 6 months in both animals, 3.1±1.3 and 6.4±0.9 out of the T cell top 10 contributing clones were highly biased towards T cells and away from all other lineages up to 17 and 23 months. In contrast to the highly biased NK contributors, these T biased clones did not extinguish in the time surveyed so far, and more frequently were detected at low levels in the My/B lineages. The degree of bias of these high contributing clones towards the NK and T lineages was steady over time, suggesting that strong clonal bias is stable. The top 10 clones in NK contributed a significantly greater fraction of hematopoiesis (20±4% and 38±10%) than the top 10 clones in the T lineage (15±2% and 13±2%) in both animals (p=0.01, p<0.0001). In all lineages at all time points from 1 to 23 months, a small subset (14±3%) of clones detected at each individual time point contributed at least 50% of barcoded hematopoiesis. Polyclonality increased following initial hematopoietic reconstitution but started to fall around 6 months, reflecting the increased contribution of certain high contributing, totipotent clones. These clones did not extinguish after beginning to contribute, and have characteristics of long-term repopulating HSCs. We observed novel instability in the lineage bias of some individual HSPC contributions. For instance, an early totipotent clone which was a top 10 producer of granulocytes halted B cell contribution almost completely at 4.5 months, before regaining it by 5 months later and becoming the highest contributing clone in the entire animal. The high degree of bias towards and away from certain lineages observed in many long term repopulating HSCs is not necessarily in violation of hypothesized progenitor hierarchies, but it indicates the presence of poorly understood nuances in clonal control. In addition to the long-term dominant unbiased HSCs whose hematopoietic contribution increased over time, we noticed a cohort of clones biased towards a combined myeloid/B cell lineage. The varied clonal patterns observed between animals could result from individual differences in transplant progenitor composition or dose, or degree of recipient endogenous HSPC depletion. Understanding the lifespan and population dynamics of repopulating clones in macaques after transplantation is relevant for optimizing human hematopoietic stem cell transplants and also provides an approach for identifying progenitor populations, inferring mechanisms of cell fate control, and calculating rates of differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals TWIST1 Preserves Hematopoietic Stem Cell Function Via Repression of Cacna1b Expression

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-12
Author(s):  
Nan Wang ◽  
Jing Yin ◽  
Na You ◽  
Dan Guo ◽  
Yangyang Zhao ◽  
...  
Keyword(s):  
Stem Cell ◽  
Steady State ◽  
Calcium Channel ◽  
Cell Fate ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem ◽  
Voltage Gated ◽  
Voltage Gated Calcium Channel

The mitochondria of hematopoietic stem cell (HSC) play crucial roles in regulating cell fate and in preserving HSC functionality and survival. However, the mechanism underlying its regulation remain poorly understood. Here, we identify transcription factor TWIST1 as a novel regulator of HSC maintenance through modulating mitochondrial function. We demonstrate that Twist1 deletion results in a significantly decreased long-term HSC (LT-HSC) frequency, markedly reduced dormancy and self-renewal capacities and skewed myeloid differentiation in steady-state hematopoiesis. Twist1-deficient LT-HSC are more compromised in tolerance of irradiation and 5 fluorouracil-induced stresses, and exhibit typical phenotypes of senescence and higher levels of DNA damage and apoptosis. Mechanistically, Twist1 deficiency upregulates the expression of voltage-gated calcium channel Cacna1b in HSC, leading to noticeable increases in mitochondrial calcium levels, biogenesis, metabolic activity and reactive oxygen species production. Suppression of voltage-gated calcium channel by a calcium channel blocker largely rescues the phenotypic and functional defects in Twist1-deleted HSCs under both steady-state and stress conditions. Collectively, our data, for the first time, characterize TWIST1 as a critical regulator of HSC function acting through CACNA1B/Ca2+/mitochondria axis, and highlight the importance of Ca2+ in HSC maintenance. These observations provide new insights into the mechanisms for the control of HSC fate. Disclosures No relevant conflicts of interest to declare.

scholarly journals Engraftment of rare, pathogenic donor hematopoietic mutations in unrelated hematopoietic stem cell transplantation

2020 ◽  
Vol 12 (526) ◽  
pp. eaax6249 ◽  
Author(s):  
Wing Hing Wong ◽  
Sima Bhatt ◽  
Kathryn Trinkaus ◽  
Iskra Pusic ◽  
Kevin Elliott ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
High Throughput Sequencing ◽  
Limit Of Detection ◽  
Cell Transplant ◽  
Younger Adults ◽  
Hematopoietic Stem ◽  
Clonal Hematopoiesis ◽  
Age Related ◽  
Over Time

Clonal hematopoiesis is associated with various age-related morbidities. Error-corrected sequencing (ECS) of human blood samples, with a limit of detection of ≥0.0001, has demonstrated that nearly every healthy individual >50 years old harbors rare hematopoietic clones below the detection limit of standard high-throughput sequencing. If these rare mutations confer survival or proliferation advantages, then the clone(s) could expand after a selective pressure such as chemotherapy, radiotherapy, or chronic immunosuppression. Given these observations and the lack of quantitative data regarding clonal hematopoiesis in adolescents and young adults, who are more likely to serve as unrelated hematopoietic stem cell donors, we completed this pilot study to determine whether younger adults harbored hematopoietic clones with pathogenic mutations, how often those clones were transferred to recipients, and what happened to these clones over time after transplantation. We performed ECS on 125 blood and marrow samples from 25 matched unrelated donors and recipients. Clonal mutations, with a median variant allele frequency of 0.00247, were found in 11 donors (44%; median, 36 years old). Of the mutated clones, 84.2% of mutations were predicted to be molecularly pathogenic and 100% engrafted in recipients. Recipients also demonstrated de novo clonal expansion within the first 100 days after hematopoietic stem cell transplant (HSCT). Given this pilot demonstration that rare, pathogenic clonal mutations are far more prevalent in younger adults than previously appreciated, and they engraft in recipients and persist over time, larger studies with longer follow-up are necessary to correlate clonal engraftment with post-HSCT morbidity.

Combined Single Cell Lineage and Transcriptome Sequencing Unveils Cell-Autonomous Regulators of Hematopoietic Stem Cell Fate

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 446-446
Author(s):  
Alejo E Rodriguez-Fraticelli ◽  
Caleb S Weinreb ◽  
Allon Moshe Klein ◽  
Shou-Wen Wang ◽  
Fernando D Camargo
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Conflicts Of Interest ◽  
Large Fraction ◽  
Cell Lineage ◽  
Gene Signature ◽  
Hematopoietic Stem ◽  
Cell State

Blood regeneration upon transplantation relies on the activity of long-term repopulating hematopoietic stem cells (LT-HSCs). One of the major controversies in hematopoiesis relates to the apparently different properties that HSCs have in transplantation versus unperturbed settings. In unperturbed steady state hematopoiesis, the most potent HSCs appear to be mostly dormant, and only producing platelet-lineage cells. In turn, upon transplant, even a single transplanted HSC can actively divide and regenerate hundreds of millions of blood progenitors of all lineages. It would thus appear that HSCs have different fundamental properties in each study system. However, most transplantation studies have only tracked the lineage output of the transplanted HSC clones, and rarely the regeneration of the HSC compartment itself. In addition, clonal assays have not been performed at sufficient resolution to fully capture the diversity and clonal complexity of the regenerated HSC compartment. Here, we have used expressible barcodes, which can be sequenced in conventional single cell RNAseq assays, to simultaneously record the functional outcomes and transcriptional states of thousands of HSCs. Our analysis revealed multiple clonal HSC behaviors following transplantation that drastically differ in their differentiation activity, lineage-bias and self-renewal. Surprisingly, we witnessed a large fraction of clones that efficiently repopulate the HSC compartment but show limited contribution to differentiated progeny. Furthermore, these inactive clones have increased competitive multilineage serial repopulating capacity, implying that shortly after transplant a subset of clones reestablishes the native-like LT-HSC behaviors. Our results also argue that this clonal distribution of labor is controlled by cell autonomous, heritable properties (i.e. the epigenetic cell state). Then, using only our clonal readouts to segregate single HSC transcriptomes, we unveiled the transcriptional signatures that associated with unique HSC outcomes (platelet bias, clonal expansion, dormancy, etc.) and unraveled, for the first time, a gene signature for functional long-term serially repopulating clones. We interrogated the drivers of this cell state using an in vivo inducible CRISPR screening and identified 5 novel regulators that are required to regenerate the HSC compartment in a cell autonomous fashion. In conclusion, we demonstrate that functional LT-HSCs share more similar properties in native and transplantation hematopoiesis than previously expected. Consequently, we unveil a definition of the essential, common functional properties of HSCs and the molecular programs that control them. Figure 1 Disclosures No relevant conflicts of interest to declare.

T(15;17)-PML/RAR-Induced Leukemogenesis: Long-Term Repopulating Hematopoietic Stem Cells as the Initial Target and More Mature Progenitors as the Potential Targets for Final Leukemic Transformation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3980-3980 ◽  
Author(s):  
Claudia Oancea ◽  
Brigitte Rüster ◽  
Jessica Roos ◽  
Afsar Ali Mian ◽  
Tatjana Micheilis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Population ◽  
Conflicts Of Interest ◽  
Cell Model ◽  
Leukemic Cell ◽  
Hematopoietic Stem ◽  
Proliferation Potential

Abstract Abstract 3980 Poster Board III-916 Stem cells have been shown to play an important role in the pathogenesis and maintenance of a significant number of malignancies, including leukemias. Similar to normal hematopoiesis the AML cell population is thought to be hierarchically organized. According to this model, only a few stem cells (LSC) are able to initiate and maintain the disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARα fusion protein. Treatment of APL with all-trans retinoic acid (t-RA) as monotherapy induces CR, but not molecular remission (CMR), followed by relapse within a few months. In contrast arsenic as monotherapy induces high rates of CR and CMR followed by a long relapse-free survival. We recently have shown that in contrast to t-RA, arsenic efficiently targets PML/RAR-positive stem cells, whereas t-RA increases their proliferation. For a better characterization of LSC in APL which has to be targeted for an efficient eradication of the disease we wanted to characterize the leukemia-initiating cell and the cell population able to maintain the disease in vivo. The model was based on a classical transduction/transplantation system of murine Sca1+/lin- HSC combined with a novel approach for the enrichment of transformed cells with long-term stem cell properties. We found that PML/RAR induced leukemia from the Sca1+/lin- HSC with a frequency of 40% and a long latency of 8-12 months independently of its capacity to increase dramatically replating efficiency and CFU-S12 potential as expression of the differentiation block and proliferation potential of derived committed progenitors. Based on the hypothesis that PML/RAR exerts its leukemogenic effects on only a small proportion of the Sca1+1/lin- population, we proceeded to select and to amplify rare PML/RAR-positive cells with the leukemia-initiating potential, by a negative selection of cell populations with proliferation potential without long term stem cell-capacity (LT). Therefore we expressed PML/RAR in Sca1+/lin- cells and enriched this population for LT- (lin-/Sca1+/c-Kit+/Flk2-) and ST-HSC (lin-/Sca1+/c-Kit+/Flk2+). After a passage first in semi-solid medium for 7 days and subsequent transplantation into lethally irradiated mice, cells from the ensuing CFU-S day12 were again transplanted into sublethally recipient mice. After 12 to 36 weeks, 6/6 mice developed acute myeloid leukemia without signs of differentiation in the group transplanted with the lin-/Sca1+/c-Kit+/Flk2- population but not from that transplanted with lin-/Sca1+/c-Kit+/Flk2+ cells. This leukemia was efficiently transplanted into secondary recipients. The primary leukemic cell population gave origin to 6 clearly distinct subpopulations defined by surface marker pattern as an expression of populations with distinct differentiation status, able - after sorting - to give leukemia in sublethally irradiated recipients: Sca1+/c-Kit+/CD34- (LT-HSC), Sca1+/c-Kit+/CD34+ (ST-HSC), Sca1-/c-Kit+, B220lo/GR1+/Mac1+, B220hi/GR1+/Mac1+, B220-/Gr1-/Mac1-. Interestingly, all leukemias from the different population presented an identical phenotype. These findings strongly suggest that there is a difference between a leukemia-initiating (L-IC) and leukemia-maintaining (L-MC) cell population in the murine PML/RAR leukemia model. In contrast to the L-IC, represented by a very rare subpopulation of primitive HSC, recalling a hierarchical stem cell model, the L-MC is represented by a larger cell population with a certain grade of phenotypical heterogeneity, but a high grade of functional homogeneity recalling a stochastic cancer induction model. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic Stem Cells Are Dependent On Homologous Recombination Only During Active Cell Cycle in Nuclease-Mutant Exonuclease1mut mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1225-1225
Author(s):  
Amar Desai ◽  
Stanton L. Gerson ◽  
Yulan Qing
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Stem Cell ◽  
Homologous Recombination ◽  
Conflicts Of Interest ◽  
Whole Body ◽  
Hematopoietic Stem ◽  
Increased Sensitivity ◽  
Quiescent Hscs

Abstract Abstract 1225 Hematopoietic stem cell (HSC) maintenance and self-renewal is crucial for long term tissue repopulation and immune function. HSC populations require functional DNA repair pathways in order to maintain their reconstitution capabilities but the pathways involved and the mechanisms of regulation are still being elucidated. It has been proposed that quiescent HSCs rely on the error prone non homologous end joining pathway for DNA double strand break (DSB) repair while HSCs in cycle use both NHEJ and the high fidelity homologous recombination (HR), but functional in vivo studies have not yet been completed. Exonuclease 1 participates in homologous recombination. We used Exo1mut fibroblasts to demonstrate that loss of Exo1 function results in a defective HR response, increased sensitivity to DSB inducing agents, and aberrant DNA damage signaling. However, Exo1mut mice did not appear to require HR to maintain quiescent HSCs at steady state or to respond to DNA damage. Exo1mutmice were able to sustain long term serial repopulation, displayed no defect in competitive repopulation or quiescence maintenance, and did not display increased sensitivity to whole body ionizing radiation (IR). In contrast, when Exo1mut HSCs were pushed into cell cycle with 5-Fluorouracil, the hematopoietic population and HSCs became hypersensitive to IR stress relative to WT B6 mice, as shown by decreased bone marrow cellularity, colony forming unit defects, loss of the HSC population, and finally animal death. Thus, loss of Exo1, and in turn fully functional HR, in quiescent HSC is not critical to stem cell function, survival, or recovery after DNA damage, whereas HR mediated repair of DNA damage is essential for HSC maintenance after cell cycle entry. In HSCs, DNA damage repair response, and sensitivity is dependent on cell cycle. Disclosures: No relevant conflicts of interest to declare.

Identification of a Developmentally-Restricted Hematopoietic Stem Cell That Gives Rise to Innate-like Lymphocytes

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4302-4302
Author(s):  
Anna E Beaudin ◽  
Scott W. Boyer ◽  
Gloria Hernandez ◽  
Camilla E Forsberg
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Lineage Tracing ◽  
Lymphoid Cells ◽  
Gfp Expression ◽  
Hematopoietic Stem

Abstract The generation of innate-like immune cells distinguishes fetal hematopoiesis from adult hematopoiesis, but the cellular mechanisms underlying differential cell production during development remain to be established. Specifically, whether differential lymphoid output arises as a consequence of discrete hematopoietic stem cell (HSC) populations present during development or whether the fetal/neonatal microenvironment is required for their production remains to be established. We recently established a Flk2/Flt3 lineage tracing mouse model wherein Flk2-driven expression of Cre recombinase results in the irreversible switching of a ubiquitous dual-color reporter from Tomato to GFP expression. Because the switch from Tom to GFP expression in this model involves an irreversible genetic excision of the Tomato gene, a GFP+ cell can never give rise to Tom+ progeny. Using this model, we have definitively demonstrated that all functional, adult HSC remain Tomato+ and therefore that all developmental precursors of adult HSC lack a history of Flk2 expression. In contrast, adoptive transfer experiments of Tom+ and GFP+ fetal liver Lin-cKit+Sca1+ (KLS) fractions demonstrated that both Tom+ and GFP+ fetal HSC support serial, long-term multilineage reconstitution (LTR) in irradiated adult recipients. We have therefore identified a novel, developmentally restricted HSC that supports long-term multilineage reconstitution upon transplantation into an adult recipient but does not normally persist into adulthood. Developmentally-restricted GFP+ HSC display greater lymphoid potential, and regenerated both innate-like B-1 lymphocytes and Vg3-expressing T lymphocytes to a greater extent than coexisting Tom+ FL and adult HSC. Interestingly, whereas developmental regulation of fetal-specific B-cell subsets appears to be regulated cell-instrinsically, as fetal HSC generated more innate-like B-cells than adult HSC even within an adult environment, T-cell development may be regulated both cell intrinsically and extrinsically, as both the cell-of-origin and the fetal microenvironment regulated the generation of innate-like T-cells. Our results provide direct evidence for a developmentally restricted HSC that gives rise to a layered immune system and describes a novel mechanism underlying the source of developmental hematopoietic waves. As early lymphoid cells play essential roles in establishing self-recognition and tolerance, these findings are critical for understanding the development of autoimmune diseases, allergies, and tolerance induction upon organ transplantation. Furthermore, by uncoupling self-renewal capacity in situ with that observed upon transplantation, our data suggests that transplantation- and/or irradiation-induced cues may allow for the engraftment of developmental HSC populations that do not normally persist in situ. As LTR upon transplantation has served as the prevailing definition of adult HSC origin during development, our data challenge the current conceptual framework of adult HSC origin. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pbx1 Regulates the Self-Renewal of Long-Term Hematopoietic Stem Cells by Maintaining Their Quiescence.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 94-94 ◽  
Author(s):  
Francesca Ficara ◽  
Mark J. Murphy ◽  
Min Lin ◽  
Michael L. Cleary
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Acute Leukemia ◽  
B Cell ◽  
Quiescent State ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cell Fate Decisions

Abstract Pbx1 is a proto-oncogene that was originally discovered at the site of chromosomal translocations in pediatric acute leukemia. It codes for a homeodomain transcription factor, which is a component of hetero-oligomeric protein complexes that regulate developmental gene expression. Lack of Pbx1 is associated with multiple patterning malformations, defects in organogenesis, and severe fetal anemia, however embryonic lethality has prevented an assessment of its roles in the adult hematopoietic stem cell (HSC) compartment and in lymphoid differentiation. The objective of this study was to characterize the physiological roles for Pbx1 in the hematopoietic system, specifically in the regulation of cell fate decisions involved in the timing and/or extent of postnatal HSC and progenitor proliferation, self-renewal or differentiation capacity. A genetic approach was employed to conditionally inactivate Pbx1 in the hematopoietic compartment in vivo using Cre recombinase expressed under the control of the Tie2 or Mx1 promoters. A crucial role for Pbx1 in the development of the lympho-hematopoietic system was evidenced by reduced size, cell number, and altered architectures of the thymus and spleen in mutant mice. A marked reduction was observed in the bone marrow (BM) pro- and pre-B cell compartment, as well as a striking reduction (up to 10-fold) in common lymphoid progenitors (CLP), suggesting a role for Pbx1 at a critical stage of lymphoid development where acute leukemia likely originates. Accordingly, abnormal T cell development was observed in the thymus. Common myeloid progenitors (CMP) and Lin-cKit+Sca1+ (LKS, enriched in HSCs) cells were also reduced, as well as long-term stem cells (LT-HSCs, reduced 7-fold on average). Assessment of the proliferation status of LT- and ST (short-term)-HSCs, as well as multi-potent progenitors (MPP), revealed that the reduction of the HSC compartment was associated with a higher number of stem cells exiting the G0 phase, thus losing their quiescent state. Strikingly, Pbx1-deficient BM cells failed to engraft in competitive transplants, but were able to reconstitute congenic recipients in the absence of competition, indicating a profound defect of functional HSCs, which nevertheless retained reconstitution potential. Importantly, Pbx1 deficient HSCs progressively disappeared from primary transplant recipients, and were unable to engraft secondary recipients, demonstrating that Pbx1 is crucial for the maintenance of LT-HSC self-renewal. Microarray studies performed on mutant and wt LT- and ST-HSCs, followed by bioinformatics analysis, showed that in the absence of Pbx1 LT-HSCs are characterized by premature expression of a large subset of ST-HSC genes. The up-regulated differentially expressed transcripts are enriched for cell cycle regulatory genes, consistent with the observed increased cycling activity. Notably, more than 8% of the down-regulated genes are related to the Tgf-beta pathway, which serves a major role in maintaining HSC quiescence. Moreover, B-cell specific genes, which are expressed in the wt LT-HSC compartment, are down-regulated in the absence of Pbx1, suggesting that the observed reduction in CLP and B-cell numbers ultimately arose from a stem cell defect in lymphoid priming. We conclude that Pbx1 is at the apex of a transcriptional cascade that controls LT-HSC quiescence and differentiation, thus allowing the maintenance of their self-renewal potential, crucial for the homeostasis of the lympho-hematopoietic system.

scholarly journals An Ex Vivo Bioengineered Human Liver Microenvironment to Support Hematopoietic Stem Cell Maintenance and Expansion

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-10
Author(s):  
Na Yoon Paik ◽  
Grace E. Brown ◽  
Lijian Shao ◽  
Kilian Sottoriva ◽  
James Hyun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Human Umbilical Cord ◽  
Hematopoietic Stem ◽  
Main Site

Over 17,000 people require bone marrow transplants annually, based on the US department of Health and Human Services (https://bloodcell.transplant.hrsa.gov). Despite its high therapeutic value in treatment of cancer and autoimmune disorders, transplant of hematopoietic stem cells (HSC) is limited by the lack of sufficient source material due primarily inadequate expansion of functional HSCs ex vivo. Hence, establishing a system to readily expand human umbilical cord blood or bone marrow HSCs in vitro would greatly support clinical efforts, and provide a readily available source of functional stem cells for transplantation. While the bone marrow is the main site of adult hematopoiesis, the fetal liver is the primary organ of hematopoiesis during embryonic development. The fetal liver is the main site of HSC expansion during hematopoietic development, furthermore the adult liver can also become a temporary extra-medullary site of hematopoiesis when the bone marrow is damaged. We have created a bioengineered micropatterned coculture (MPCC) system that consists of primary human hepatocytes (PHHs) islands surrounded and supported by 3T3-J2 mouse embryonic fibroblasts. Long-term establishment of stable PHH-MPCC allows us to culture and expand HSC in serum-free medium supplemented with pro-hematopoietic cytokines such as stem cell factor (SCF) and thrombopoietin (TPO). HSCs cultured on this PHH-MPCC microenvironment for two weeks expanded over 200-fold and formed tight clusters around the periphery of the PHH islands. These expanded cells also retained the expression of progenitor markers of Lin-, Sca1+, cKit+, as well as the long-term HSC phenotypic markers of CD48- and CD150+. In addition to the phenotypic analysis, the expanded cells were transplanted into lethally irradiated recipient mice to determine HSC functionality. The expanded cells from the PHH-MPCC microenvironment were able to provide multi-lineage reconstitution potential in primary and secondary transplants. With our bioengineered MPCC system, we further plan to scale up functional expansion of human HSC ex vivo and to better understand the mechanistic, cell-based niche factors that lead to maintenance and expansion HSC. Disclosures No relevant conflicts of interest to declare.

TIMP-1 Deficiency Subverts Cell Cycle Dynamics in Long-Term HSCs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2514-2514
Author(s):  
Lara Rossi ◽  
Margaret Goodell
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Conflicts Of Interest ◽  
Biological Behavior ◽  
Cell Cycle Distribution ◽  
Matrix Remodeling ◽  
Hematopoietic Stem ◽  
Stem Cell Quiescence

Abstract Abstract 2514 Poster Board II-491 In addition to the consolidated role in extracellular matrix remodeling, the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) has been suggested to be involved in the regulation of numerous biological functions, including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of hematopoietic stem cells (HSCs), whose biological behavior is the synthesis of both microenvironmental and intrinsic cues. We found that TIMP-1−/− mice have decreased HSC numbers and, consistent with this finding, TIMP-1−/− HSCs display reduced capability of long-term repopulation. Interestingly, the cell cycle distribution of TIMP-1−/− LT-HSCs is profoundly distorted, with a consistent proportion of the stem cell pool arrested in the G1 phase, suggesting that TIMP-1 is intrinsically involved in the regulation of the HSC proliferation dynamics. Indeed, HSCs exhibit a higher proliferation rate, leading to an increased formation of CFU-C in vitro and spleen colonies (CFU-S) after transplant. Of note, TIMP-1−/− HSCs present decreased levels of CD44 glycoprotein, whose expression has been proven to be controlled by p53, the master regulator of the G1/S transition. Our findings establish TIMP-1 role in HSC function, suggesting a novel mechanism presiding over stem cell quiescence and potentially involved in the development of hematological diseases. Disclosures: No relevant conflicts of interest to declare.

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