preparation of enriched live cell suspension for nuclei isolation from ipsc-derived blood cells

גרעינים באיכות גבוהה מתאים המטופויטיים שמקורם ב-iPSC למחקרים מולטיומיים

Hematopoiesis is a relatively well-characterized developmental system, but an inability to recapitulate blood cell formation in vitro demonstrates an incomplete understanding of related factors. Induced pluripotent stem cell (iPSC)-based hematopoiesis models can help elucidate key developmental factors and related biology. The iPSC system also offers an excellent model to study blood disorders, and iPSC-based blood cells have been developed to produce translationally and therapeutically relevant reagents1,2,3,4. Single-cell studies have been used extensively to investigate iPSC-based developmental biology, including cell types that are produced during hematopoiesis5. Compared to bulk RNA sequencing, which cannot infer relationships between cell subpopulations6, single-cell modalities allow for assessments of cell heterogeneity and can facilitate the identification and characterization of cell development6,7.
The formation of hematopoietic cells from iPSCs requires sequential differentiation through primitive streak, mesoderm, and endothelial states to form hemogenic endothelial cells. Hemogenic endothelial cells are direct precursors to hematopoietic stem and progenitor cells8. These cell types have remarkably different morphological and cellular properties. There is a limited understanding of the epigenetic landscape and developmental dynamics of in vitro-derived hematopoietic cell models, though this is essential knowledge to unlock the full therapeutic potential of the iPSC system. In many cases, only single cell analysis modalities allow for the identification of cell populations, transcriptional activities, chromatin landscapes, and regulatory mechanisms that govern development among heterogenous cell preparations.
Two methodologies, single-cell RNA sequencing (scRNAseq) and single nuclei RNA sequencing (snRNAseq), can reveal transcriptional insights at the individual cell level9. A principal factor dictating data validity and reliability is the uncompromising need for samples that meet stringent quality criteria. These include precise requirements for cell/nuclear density, optimal viability, and minimal clumping. Preparation of single nuclei can be technically challenging. There can be additional challenges associated with the use of previously cryopreserved cells.
We note four important potential limitations to standard scRNAseq approaches. First, standard protocols for generating cell suspensions often reference preparations from fresh cells or tissues, rather than those retrieved post-cryopreservation10. This can curtail the utility of potentially valuable resources. Second, the dissociation efficiency of diverse cell types is variable. For instance, many immune cell types undergo dissociation with relative ease. In contrast, stromal cells, fibroblasts, and endothelial cells, which are normally embedded in the extracellular matrix and basement membrane, have unique cell properties which can complicate nuclei isolation11,12. These properties can necessitate aggressive dissociation protocols, which may jeopardize the structural integrity of more delicate cell populations. Third, some cells (e.g., megakaryocytes) can surpass 100 &#181;m in diameter and pose difficulties for several existing commercial single-cell platforms13. Additionally, improper handling can lead to cellular damage and stress responses during the initial steps of cell isolation, involving enzymatic hydrolysis and mechanical dissociation, which can impact gene expression profiles11,14.
For these and other reasons, a single nucleus approach may be a preferable alternative to single cell methods15. Given the superior stability of the nuclear membrane compared to the cell membrane, the nuclear envelope may remain intact even after tissue cryopreservation16. This can facilitate nuclear extraction and enhance the diversity of sample types suitable for next generation sequencing modalities. Second, nuclei exhibit heightened resistance to mechanical perturbations and tend to manifest fewer transcriptional shifts during dissociation14. Therefore, nuclei can provide greater RNA stability and more reproducible transcriptional profiles. A third noteworthy advantage of single nuclei methods is a lack of cell size constraints and an applicability for larger cells, like cardiomyocytes or megakaryocytes12. Fourth, nuclei serve as suitable substrates for multiomics analyses, which offer expanded insights from integrated analysis of gene expression, accessible chromatin regions, and other parameters17, enabling deeper understanding of cell heterogeneity, development, and functional states18.
By profiling iPSCs during hematopoietic development, multiomics approaches can help define developmental processes in normal or pathologic disease models. Comparisons of iPSC-derived cells to primary cells or tissues can also provide an assessment of iPSC model fidelity to in vivo biology, facilitating iPSC model improvement and the discovery of novel cell populations and factors driving blood formation.
Although many groups have successfully navigated nuclear isolation and resultant next generation sequencing analysis, isolating high quality nuclei remains a barrier to some laboratories interested in performing single nucleus-based analyses. This manuscript details a protocol for isolating quality nuclei from previously cryopreserved iPSC-derived cells. We have focused on adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structure and content that demand tailored modifications and troubleshooting to heighten recovery of high-quality nuclei amenable for next generation sequencing or other experiments.

מחבר זרקור: התקדמות בייצור תאי דם חוץ גופיים באמצעות מולטיומיקה חד-תאית וגנומיקה פונקציונלית

בידוד גרעיני מתאי דם שמקורם בהקפאה חוץ גופית

My lab studies genes and mechanisms that affect blood cell formation. We produce blood cells from human-induced pluripotent stem cells, and we wanna do this more efficiently to support clinical-scale production of cell therapies that are tailored for specific patients. We use single cell genomics modalities to identify patterns in gene expression and other factors that can help support blood cell formation.One major challenge for in vitro blood cell production is that is expensive and inefficient. We are using genetic approaches to address this. We want to use clues from human genetics and in vivo hematopoiesis to guide the improvements that we make to the in vitro system.We're trying to use functional genomics, and single nucleus analyses to advance the field in terms of identifying genes and signaling networks that drive the formation of the right types of blood cells in vitro. Creating high quality multi-omic data set has helped us to discover transcription factors, energy, and regular machinery that drive in vitro differentiation of blood cells. We are using this data to understand human genetic data like loci, and uncertain factors that are driving differentiation that we haven't been able to see before.We're very excited to build on insights from these multimodal data sets that we've been able to generate based on the isolation of high quality nuclei. We intend to focus our future studies on genes and developmental mechanisms that can produce blood cell types, ultimately at clinical scale in vitro.

הכנת תרחיף תאים חיים מועשר לבידוד גרעינים מתאי דם שמקורם ב-iPSC

כדי להתחיל, שלפו את בקבוקון הקריו עם תאים מחנקן נוזלי והפשירו אותו באמבט מים של 37 מעלות צלזיוס למשך שתי דקות. לאחר העברת התאים המופשרים לצינור של 15 מיליליטר, מוסיפים בהדרגה 10 מיליליטר של תווך שחומם מראש וצנטריפוגה את הצינור. השליכו את הסופרנאטנט והשהו מחדש את התאים במיליליטר אחד של תוספת DPBS המכילה FBS וסידן כלורי.לאחר מכן, ערבבו מיליליטר אחד של תרחיף התא עם 50 מיקרוליטר כל אחד מקוקטייל הסרת התאים המתים וקוקטייל בחירת הביוטין בצינור פוליסטירן עגול בעל תחתית עגולה של חמישה מיליליטר ודגירה. לאחר המערבולת, מוסיפים 100 מיקרוליטר של חרוזי דקסטרן לתרחיף התא, ומערבבים בעדינות פעמיים עם הפיפטה. לאחר מכן הוסיפו 1.3 מיליליטר של DPBS בתוספת לתערובת ודגרו עם מגנט במשך שלוש דקות בטמפרטורת החדר.הפוך את המגנט והצינור כדי לשפוך את תרחיף התא החי לתוך צינור חרוטי חדש של 15 מיליליטר. לדחוף את התאים בצנטריפוגה. לאחר הסרת רוב supernatant, resuspend את התאים במיליליטר אחד של DPBS המכיל 0.04% אלבומין בסרום בקר.

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Research

JoVE Journal

Developmental Biology

Preparation of Enriched Live Cell Suspension for Nuclei Isolation from iPSC-Derived Blood Cells

75 Views.  Children’s Hospital of Philadelphia. כדי להתחיל, שלפו את בקבוקון הקריו עם תאים מחנקן נוזלי והפשירו אותו באמבט מים של 37 מעלות צלזיוס למשך שתי דקות. לאחר העברת התאים המופשרים לצינור של 15 מיליליטר, מוסיפים בהדרגה 10 מיליליטר של תווך שחומם מראש וצנטריפוגה את הצינור. השליכו את הסופרנאטנט והשהו מחדש את התאים במיליליט...

Video: הכנת תרחיף תאים חיים מועשר לבידוד גרעינים מתאי דם שמקורם ב-iPSC

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational utility as clinical testing reagents and transfusable cell therapeutics. The advent and expansion of multiomics analysis, including but not limited to single nucleus RNA sequencing (snRNAseq) and Assay for Transposase-Accessible Chromatin sequencing (snATACseq), offers the potential to revolutionize our understanding of cell development. This includes developmental biology using in vitro hematopoietic models. However, it can be technically challenging to isolate intact nuclei from cultured or primary cells. Different cell types often require tailored nuclear preparations depending on cellular rigidity and content. These technical difficulties can limit data quality and act as a barrier to investigators interested in pursuing multiomics studies. Specimen cryopreservation is often necessary due to limitations with cell collection and/or processing, and frozen samples can present additional technical challenges for intact nuclear isolation. In this manuscript, we provide a detailed method to isolate high-quality nuclei from iPSC-derived cells at different stages of in vitro hematopoietic development for use in single-nucleus multiomics workflows. We have focused the method development on the isolation of nuclei from iPSC-derived adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structural and cellular identity. The described troubleshooting steps limited nuclear clumping and debris, allowing the recovery of nuclei in sufficient quantity and quality for downstream analyses. Similar methods may be adapted to isolate nuclei from other cryopreserved cell types.

We describe a protocol for extracting high-quality nuclei from cryopreserved induced pluripotent stem cell-derived stromal/endothelial and blood cell types to support single-nucleus next-generation sequencing analyses. Producing high-quality, intact nuclei is imperative for multiomics experiments but can be a barrier to entry in the field for some laboratories.

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational ...