isolating nuclei from ipsc-derived cells for advanced multiomics research

मल्टीओमिक्स अध्ययन के लिए आईपीएससी-व्युत्पन्न हेमटोपोइएटिक कोशिकाओं से उच्च गुणवत्ता वाले नाभिक

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.

लेखक स्पॉटलाइट: सिंगल-सेल मल्टीओमिक्स और फंक्शनल जीनोमिक्स के साथ इन विट्रो ब्लड सेल प्रोडक्शन में आगे बढ़ना

क्रायोप्रेज़र्व्ड इन विट्रो व्युत्पन्न रक्त कोशिकाओं से परमाणु अलगाव

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.

उन्नत मल्टीओमिक्स अनुसंधान के लिए IPSC व्युत्पन्न कोशिकाओं से नाभिक को अलग करना

शुरू करने के लिए, एक शंक्वाकार ट्यूब में समृद्ध लाइव आईपीएससी-व्युत्पन्न सेल निलंबन प्राप्त करें और इसे चार डिग्री सेल्सियस पर पांच मिनट के लिए 460 ग्राम पर अपकेंद्रित्र करें। सतह पर तैरनेवाला त्यागने के बाद, ताजा तैयार 0.5X ठंड lysis बफर के 100 माइक्रोलीटर जोड़ें. एक P100 का प्रयोग, विंदुक ऊपर और नीचे 10 बार कोशिकाओं मिश्रण और तीन मिनट के लिए बर्फ पर ट्यूब सेते हैं.अगला, lysate ठंडा धोने बफर के 500 microliters जोड़ें, ट्यूब अपकेंद्रित्र, और सतह पर तैरनेवाला त्यागें. धोने को दोहराने के बाद, ठंडा पतला नाभिक बफर के 120 माइक्रोलीटर में गोली को फिर से निलंबित करें। 40-माइक्रोमीटर सेल स्ट्रेनर का उपयोग करके, सेल सस्पेंशन को फ़िल्टर करें और छानना में 0.4% ट्रिपैन ब्लू जोड़ें।अंत में, एक खुर्दबीन के तहत पृथक नाभिक की गुणवत्ता का आकलन करें। इस तकनीक का उपयोग करके, उच्च गुणवत्ता वाले नाभिक क्रायोप्रेज़र्ड मानव आईपीएससी-व्युत्पन्न हेमटोपोइएटिक पूर्वज कोशिकाओं से अलग किए गए थे।

isolating-nuclei-from-ipsc-derived-cells-for-advanced-multiomics

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JoVE Journal

Developmental Biology

Isolating Nuclei from iPSC-Derived Cells for Advanced Multiomics Research

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 ...