isolating nuclei from ipsc-derived cells for advanced multiomics research

멀티오믹스 연구를 위한 iPSC 유래 조혈 세포의 고품질 핵

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.

저자 스포트라이트: Advancing In Vitro Blood Cell Production with Single-Cell Multiomics and Functional Genomics(단세포 다중체역학 및 기능유전체학을 통한 체외 혈액 세포 생산의 발전)

Cryopreserved In Vitro 유래 혈액 세포로부터 핵 분리

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.

고급 멀티오믹스 연구를 위한 iPSC 유래 세포에서 핵 분리

먼저 원뿔형 튜브에서 농축된 살아있는 iPSC 유래 세포 현탁액을 얻고 섭씨 4도에서 5분 동안 460g으로 원심분리합니다. 상층액을 버린 후 갓 준비한 100마이크로리터의 0.5X 냉간 용해 완충액을 추가합니다. P100을 사용하여 피펫팅을 위아래로 10회 수행하여 세포를 혼합하고 튜브를 얼음 위에서 3분 동안 배양합니다.다음으로, 500마이크로리터의 냉각 세척 완충액을 용해물에 넣고 튜브를 원심분리한 다음 상층액을 버립니다. 세척을 반복한 후 펠릿을 120마이크로리터의 냉각된 희석된 핵 완충액에 재현탁시킵니다. 40마이크로미터 세포 여과기를 사용하여 세포 현탁액을 여과하고 여과액에 0.4% 트리판 블루를 추가합니다.마지막으로, 현미경으로 분리된 핵의 품질을 평가합니다. 이 기술을 사용하여 냉동 보존된 인간 iPSC 유래 조혈 전구 세포에서 고품질 핵을 분리했습니다.

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

Research

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 ...