Culture Of Human Hematopoietic Stem Cells Research Articles

Ex vivo reprogramming of human hematopoietic stem cells is accompanied by increased transcripts of genes regulating metabolic integrity

The regenerative potential of human hematopoietic stem cells (HSCs) is functionally defined by their ability to provide life-long blood cell production and to repopulate myeloablated allogeneic transplant recipients. The expansion of HSC numbers is dependent not only on HSC divisions but also on a coordinated adaptation of HSCs to metabolic stress. These variables are especially critical during the ex vivo culture of HSCs with cytokine combinations, which frequently results in HSC exhaustion. We have previously reported that human CD34+ hematopoietic stem and progenitor cells (HSPCs) can be efficiently reprogrammed ex vivo and that the number of phenotypic HSCs with long-term repopulation capacity is expanded in the presence of a combination of cytokines and an epigenetic modifier. Here, we present evidence that ex vivo HSC reprogramming and maintenance is accompanied by increased transcripts of genes regulating metabolic integrity, including SIRT1 and SIRT3.

Modeling Premalignant Transformation of Hematopoietic Stem Cells in a Nanobioreactor in Microgravity

Microgravity coupled with increased radiation exposure aboard the ISS provides a unique environment to simulate and study response to injury, inflammatory signaling, aging, and (pre-)malignant transformation of normal human hematopoietic stem cells (HSCs) in an accelerated timeframe. The NASA Twin study suggests that genomic, epigenomic, epitranscriptomic, and proteomic changes may detrimentally impact hematopoietic stem and immune cell fitness and induce stem cell exhaustion (Garrett-Bakelman et al., Science, 2019). Moreover, changes indicative of pre-cancer stem cell generation, such as increased chromosome translocations and inversions, occurred and persisted post-flight. Additionally, a recent publication entitled Multisystem Toxicity in Cancer: Lessons from NASA's Countermeasures Program found significant similarities between the multisystem physiological toxicities experienced in cancer patients as well as during spaceflight (Scott et al, Cell 2019). These reports highlight the benefits of studying injury response, changes in mutational profiles and cancer evolution in microgravity at the stem cell level. For this study, we designed a novel bioreactor system to support the culture of donor-derived human HSCs in low Earth orbit (LEO). A sponge matrix and stromal cells model the microenvironment HSCs reside in within the bone marrow niche. Testing on Earth confirmed our system's ability to maintain stem cell fitness over several weeks. To assess stem cell physiology in LEO over time, we lentivirally transduced a reporter into the HSCs pre-flight (Pineda et al., Scientific Reports 2016), which allows for cell cycle tracking via fluorescence imaging. This will provide data for assessment of stem cell health, maintenance and functionality. Furthermore, we are analyzing cytokine profiles and mutational status post-flight, with a focus on signatures we have previously connected to (pre-)malignant transformation via RNA sequencing analysis (Jiang, Cancer Cell 2019). The first batch of bioreactors launched with the SpX-24 mission in December 2021 and the second batch is currently on orbit as part of the SpX-25 mission. This investigation has the potential to provide valuable insights into the maintenance of hematopoietic stem cell health and functionality, HSC response to injury through accumulation of mutations and, eventually, the mechanisms fueling long-term (pre-)malignant transformation into leukemia stem cells.

Abstract 3154: Assessing hematopoietic stem cell fitness within a nanobioreactor in microgravity

Abstract Microgravity coupled with increased radiation exposure aboard the ISS provides a unique environment to simulate and study response to injury, inflammatory signaling, aging, and (pre-)malignant transformation of normal human hematopoietic stem cells (HSCs) in an accelerated timeframe. The NASA Twins study suggests that genomic, epigenomic, epitranscriptomic, and proteomic changes may detrimentally impact hematopoietic stem and immune cell fitness and induce stem cell exhaustion (Garrett-Bakelman et al., Science, 2019). Moreover, changes indicative of pre-cancer stem cell generation such as increased chromosome translocations and inversions occurred and persisted post-flight. Additionally, a recent publication entitled Multisystem Toxicity in Cancer: Lessons from NASA’s Countermeasures Program found significant similarities between the multisystem physiological toxicities in cancer patients and during spaceflight (Scott et al, Cell 2019). These reports highlight the benefits of studying injury response, changes in mutational profiles and cancer evolution in microgravity at the stem cell level. For this study, we designed a novel bioreactor system to support the culture of donor-derived human HSCs in low Earth orbit (LEO). A sponge matrix and stromal cells model the microenvironment HSCs reside in within the bone marrow niche. Testing on Earth confirmed our system’s ability to maintain stem cell fitness over several weeks. To assess stem cell physiology in LEO over time, we lentivirally transduce a reporter into the HSCs pre-flight (Pineda et al., Scientific Reports 2016), which allows for cell cycle tracking via fluorescence imaging. This will provide data for assessment of stem cell health, maintenance and functionality. Furthermore, we will be analyzing mutational status post-flight, with a focus on signatures we have previously connected to (pre-)malignant transformation via RNA sequencing analysis (Jiang, Cancer Cell 2019). Our bioreactors are scheduled to launch as part of the SpaceX CRS-24 mission on Dec 21, 2021. This investigation may provide valuable insights into the maintenance of hematopoietic stem cell health and functionality, response to injury through accumulation of mutations and, eventually, the mechanisms fueling long-term (pre-)malignant transformation into leukemia stem cells. Citation Format: Luisa Ladel, Jessica Pham, Isabelle Oliver, Larisa Balaian, Catriona HM Jamieson. Assessing hematopoietic stem cell fitness within a nanobioreactor in microgravity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3154.

UM171 Preserves Epigenetic Marks that Are Reduced in Ex Vivo Culture of Human HSCs via Potentiation of the CLR3-KBTBD4 Complex.

Human hematopoietic stem cells (HSCs) exhibit attrition of their self-renewal capacity when cultured exvivo, a process that is partially reversed upon treatment with epigenetic modifiers, most notably inhibitors of histone deacetylases (HDACs) or lysine-specific demethylase LSD1. A recent study showed that the human HSC self-renewal agonist UM171 modulates the CoREST complex, leading to LSD1 degradation, whose inhibition mimics the activity of UM171. The mechanism underlying the UM171-mediated loss of CoREST function remains undetermined. We now report that UM171 potentiates the activity of a CULLIN3-E3 ubiquitin ligase (CRL3) complex whose target specificity is dictated by the poorly characterized Kelch/BTB domain protein KBTBD4. CRL3KBTBD4 targets components of the LSD1/RCOR1 corepressor complex for proteasomal degradation, hence re-establishing H3K4me2 and H3K27ac epigenetic marks, which are rapidly decreased upon exvivo culture of human HSCs.

Antagonizing Retinoic Acid Receptors Increases Myeloid Cell Production by Cultured Human Hematopoietic Stem Cells

Activities of the retinoic acid receptor (RAR)α and RARγ are important to hematopoiesis. Here, we have investigated the effects of receptor selective agonists and antagonists on the primitive human hematopoietic cell lines KG1 and NB-4 and purified normal human hematopoietic stem cells (HSCs). Agonizing RARα (by AGN195183) was effective in driving neutrophil differentiation of NB-4 cells and this agonist synergized with a low amount (10 nM) of 1α,25-dihydroxyvitamin D3 to drive monocyte differentiation of NB-4 and KG1 cells. Treatment of cultures of human HSCs (supplemented with stem cell factor ± interleukin 3) with an antagonist of all RARs (AGN194310) or of RARα (AGN196996) prolonged the lifespan of cultures, up to 55 days, and increased the production of neutrophils and monocytes. Slowing down of cell differentiation was not observed, and instead, hematopoietic stem and progenitor cells had expanded in number. Antagonism of RARγ (by AGN205728) did not affect cultures of HSCs. Studies of CV-1 and LNCaP cells transfected with RAR expression vectors and a reporter vector revealed that RARγ and RARβ are activated by sub-nM all-trans retinoic acid (EC50–0.3 nM): ~50-fold more is required for activation of RARα (EC50–16 nM). These findings further support the notion that the balance of expression and activity of RARα and RARγ are important to hematopoietic stem and progenitor cell expansion and differentiation.

The TLR1/2 agonist PAM3CSK4 instructs commitment of human hematopoietic stem cells to a myeloid cell fate

Toll-like receptors (TLRs) constitute a family of nonpolymorphic receptors that are devoted to pathogen recognition. In this work, we have explored the impact of TLR ligands (TLR-L) on human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). We show that HSCs and HPCs have a comparable pattern of expression of TLR transcripts characterized by the predominance of TLR1, -2, -3, -4 and -6. In long-term cultures of HSCs, HPCs and stromal cells, most TLR-L profoundly inhibited B-cell development while preserving or enhancing the production of myeloid cells. In short-term cultures, the TLR1/2 ligand PAM(3)CSK(4) induced a large proportion of HPCs to express markers of the myelomonocytic lineage. PAM(3)CSK(4) induced only marginal expression of myeloid lineage markers on HSCs but promoted their myeloid commitment as revealed by their acquisition of the phenotype of multi- and bipotential myeloid progenitors and by upregulation of the transcription factors PU.1, C/EBPalpha and GATA-1. Our results suggest that TLR agonists can bias the lineage commitment of human HSCs and shift the differentiation of lineage-committed progenitors to favor myelopoiesis at the expense of lymphoid B-cell development.

In Vitro T Cell Differentiation from Human Hematopoietic Stem Cells (HSC) and Lymphoid Progenitors.

The study of human thymopoiesis has been hampered by our inability to provide all the signals of the normal thymic microenvironment to human stem and progenitor cells in vitro. Although T cell production from CD34+ progenitors can be achieved in vitro, purified populations of the immature CD34+CD38- cells (which include HSC) do not generate T cells reliably with the existing assays. The most commonly cited system for human T cell assay, Fetal thymic organ culture (FTOC) from immune deficient mice, is logistically very difficult and low seeding efficiency precludes its use for clonal assays. Recently, a murine bone marrow stromal line engineered to constitutively express Delta-1 ligand (OP9-DL1 stroma), has been shown to support production of mature murine T cells from HSC [Schmitt, 2002] and ES cells [Schmitt, 2004]. Here we report the use of modifications to the OP9-DL1 system that allow assay of human T lymphopoiesis from HSC and progenitors. CD34+lin-CD38- cells (HSC) from cord blood (CB) and bone marrow (BM)cultured on OP9 control stroma (that does not express DL) produced CD19+ B cells and CD56+ NK cells and maintained CD34+ cells for several weeks. However, T cell commitment was not evident in these control cultures. In contrast, culture of HSC on OP9-DL1 stroma (Dr. Zuniga-Pflucker) resulted in robust growth of T cell precursors (as shown by expression of CD7 and cytoplasmic (cy) CD3 in over 50% of cells) and mRNA for pre-Tα. NK cells were also increased on OP9-DL1 relative to control OP9 stroma but B cell production was lost. Although T cell commitment to the CD34+CD7+ and CD34-cyCD3+CD7+ cells stages was accomplished on OP9-DL1 stroma, markers of further T cell maturation such as surface CD3, CD4 and CD8, were absent on the cells produced. Multiple combinations of growth factors (IL-7, IL-2, IL-15, IL-3, ckit ligand (KL) and thrombopoietin (Tpo)) to the OP9-DL1 stromal cultures failed to achieve further T cell maturation, although the combination of KL, Tpo, IL-7 produced optimal cell growth. However, when conditioned medium collected from human thymic stromal cultures (thyCM) (medium collected from adherent cells derived from human postnatal thymus and filtered through 0.45u) was added to OP9-DL1 co-cultures, cyCD3+ cells were increased and differentiation of CD3+CD4+ cells was achieved. Thymic CD34+lin-CD7+ cells produced large numbers of CD3+CD4+CD8+, CD4+CD8- and CD4-CD8+ cells in this system. We have now used the OP9-DL1/thyCM system to demonstrate the T cell potential of CB and BM CD34+CD38-lin-CD7- (HSC) and CB CD34+CD38-lin-CD7+ (CLP). In addition, efficient cloning of single CB HSC and primitive thymic progenitors has been achieved using this culture system. The data suggests that, although DL-1 is required and sufficient for early T lymphoid commitment, other factor(s) specific to human thymic stroma are required for production of more mature T cells; these factors can now be identified using the OP9-DL1 system. The availability of a simple monolayer culture system that allows T lymphoid commitment from HSC and progenitors and further differentiation to mature T cells will be of great value in the study of the lineage potential of progenitor populations and the regulation of human thymopoiesis.