Name: 저자 스포트라이트: 재생 의학을 위한 인간 유도 만능 줄기 세포의 간세포 정제 발전
Uploaded: 2023-12-01T12:50:00
Description: Watch this Scientific Journal Video about Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells at JoVE.com

이 작업은 문부과학성 보조금 번호[23390072]의 지원을 받았습니다.

이 연구는 상업적으로 획득한 인간 iPS 세포(253G1 균주)를 사용했습니다( 재료 표 참조).
1. 인간 iPS 세포의 유지
<ol><li>0.25 μg/cm2 iMatrix-511로 코팅된 배양 접시의 AK02 배지에서 인간 iPS 세포를 공급이 없는 상태로 유지합니다( 재료 표 참조).</li>
</ol>2. 2D 배양에서 인간 iPS 세포의 간 분화
<p class="jove_con...

Mitochondrial-ALCAM 분류에 의한 인간 iPSC 유래 간세포 정제

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule-driven+hepatocyte+differentiation+of+human+pluripotent+stem+cells.">Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells.</a> Stem Cell Reports. 4 (5), 939-952 (2015).</li><li>Hirata, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALCAM+(CD166)+is+a+surface+marker+for+early+murine+cardiomyocytes.">ALCAM (CD166) is a surface marker for early murine cardiomyocytes.</a> Cells, Tissues, Organs. 184 (3-4), 172-180 (2006).</li><li>Tohyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+metabolic+flow+enables+large-scale+purification+of+mouse+and+human+pluripotent+stem+cell-derived+cardiomyocytes.">Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes.</a> Cell Stem Cell. 12 (1), 127-137 (2013).</li></ol>

영양소 대사와 해독 작용을 하기 때문에, 간세포는 다른 세포 유형에 비해 상대적으로 많은 수의 미토콘드리아를 보유하고있다 15. ALCAM은 면역글로불린 슈퍼패밀리의 구성원이며 세포 접착 및 이동에 중요한 역할을 합니다. 간세포, 상피세포, 림프구세포, 골수세포, 섬유아세포, 신경세포 등 다양한 세포 유형에서 발현된다16. 미토콘드리아 기반 방법과 ALCAM 항?...

배아 및 유도 만능 줄기 세포(각각 ES 및 iPS)는 재생 요법을 위한 유망한 세포 공급원으로 간주됩니다. 그러나, 이러한 세포를 특정 표적 세포 유형으로 분화하는 효율성은 동일한 세포주, 프로토콜 및 실험자 1,2,3,4를 사용하는 경우에도 다를 수 있습니다. 이러한 변동성은 인간 ES/iPS 세포가 환경에 민감하게 반응하기 때문일 수 있습니다. 따라서 현재로서는 순수한 타겟 세포를 지속적으로 획득하기 어렵습니다. 안전성이 높은 재생의학을 달성하기 위해서는 치료세포에서 증식세포와 미분화줄기세포를 제거하는 것이 중요하며, 표적세포에 대한 첨단 정제 기술이 필수적이다 5,6,7.
세포 분류기는 개별 세포를 즉시 분석하고 형광 신호 강도를 기반으로 관심 있는 활성 세포를 분류하는 장치로, 유망한 솔루션을 제공합니다. 이는 세포 유형별 표면 마커의 항체 염색을 통해 또는 세포 유형별 리포터 유전자 발현을 활용하여 달성할 수 있습니다. 이 기술을 사용하여 만능 줄기 세포 유래 심근 세포 8,9,10 및 간세포11,12를 정제하는 방법에 대한 여러 보고서가 있습니다. Hattori et al.은 세포 분류기13을 사용하여 혁신적인 미토콘드리아 정제 방법을 개발했습니다. 심근세포는 미토콘드리아 활동을 통해 많은 에너지를 필요로 한다는 사실을 이용하여 살아있는 미토콘드리아 표시 염료인 TMRM(테트라메틸로다민 메틸 에스테르)으로 세포를 염색하는 데 사용하여 다양한 세포 유형을 포함하는 인간 ES 세포 유래 배아체의 FACS로 심근세포를 라벨링하고 고도로 정제할 수 있습니다. 종양원성의 부재는 정제된 심근세포를 사용한 기형종 형성 분석에 의해 확인되었습니다. 또한, Yamashita 등은 미토콘드리아 활성이 높고 ALCAM 양성 발현이 가능한 분획을 분리하여 인간 ES 세포 유래 배아체에서 간세포를 정제하는 방법을 예기치 않게 발견했습니다14. 이 방법의 근거는 간세포는 영양 대사 및 해독을 위해 ATP를 많이 소비하기 때문에 상대적으로 많은 수의 미토콘드리아를 가지고 있으며15 간세포는 세포 접착 및 이동에 중요한 역할을 하는 면역글로불린 슈퍼패밀리의 구성원인 ALCAM을 발현한다는 것입니다16.
만능줄기세포 유래 간세포에 대한 이전의 고도의 정제 방법은 유전자 변형이 필요했고, 비유전자 정제 방법은 효율이 낮았다17. 미토콘드리아 비유전적 방법은 고순도를 달성하는 장점이 있습니다. 간 전구 세포가 필요한 경우, CD133 및 CD13 또는 Dlk1 기반 방법18,19를 선택할 수 있습니다. 게놈 편집 기술의 정확도가 향상되었지만, 예상치 못한 게놈 변화(예: 발암)의 잠재적 위험을 완전히 제거할 수는 없습니다. 유전자 변형을 포함하지 않고 미토콘드리아 활성에 기반한 방법은 이러한 위험에서 자유로울 수 있습니다.
신생아 쥐의 심근세포에서 다양한 미토콘드리아 지표를 조사한 결과, TMRM은 24시간 이내에 완전히 사라지는 것으로 관찰된 반면, 다른 염료는 최소 5일 동안 남아 있었다13. 또한, TMRM과 JC-1은 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) 분석으로 평가했을 때 세포 생존율에 영향을 미치지 않은 반면, 다른 염료는 세포 생존율에 다양한 영향을 미쳤다는 점에 유의해야 한다13. TMRM은 더 높은 안전성을 보여줍니다.
현재까지 여러 2차원(2D) 분화 방법이 3차원(3D) 배아 몸체 형성에 비해 분화를 유도하기 위한 보다 효율적인 접근법으로 개발되었습니다. 이는 단계적 분화 유도 화합물 또는 사이토카인을 3D 공간이 아닌 2D 평면의 세포에 균일하게 투여할 수 있기 때문입니다. 이 연구에서, 이전에 보고된 방법20 은 인간 iPS 세포 유래 간세포로의 분화를 유도하도록 수정되었다. 여기에서는 인간 iPS 세포에서 유래한 간세포의 최신 2D 분화 및 정제를 위한 절차에 대해 자세히 설명합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>253G1 human iPS cell line</td>
			<td>RIKEN BioResource Research Center</td>
			<td>HPS0002</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>163-20145</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A Solution, Human, Recombinant</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>18585</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Nuclei Antibody, clone 235-1</td>
			<td>Chemicon</td>
			<td>MAB1281</td>
			<td>Antibody against human nuclear antigen</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50x), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>MedChemExpress</td>
			<td>HY-10182&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ciclosporin A</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>035-18961</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>034-22363</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Gel-like basement membrane matrix</td>
		</tr>
		<tr>
			<td>CultureSure Y-27632</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>036-24023</td>
			<td>ROCK inhibitor</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>047-18863</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>047-29353</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10036</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>51820-500</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Dipeptide L-Alanyl-L-Glutamine</td>
		</tr>
		<tr>
			<td>Human/Mouse/Rat/Canine ALCAM/CD166 Antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF1172</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone 21-hemisuccinate sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H2270</td>
			<td></td>
		</tr>
		<tr>
			<td>iMatrix-511 silk</td>
			<td>Nippi</td>
			<td>892021</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmunoBlock</td>
			<td>KAC</td>
			<td>CTKN001</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>ITS-G Supplement(&#215;100)</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>090-06741</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>128-06075</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Hexanoic-Try-Ile-(6)-amino Hexanoic amide (Dihexa)</td>
			<td>Toronto Research Chemicals</td>
			<td>H293745</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal Rabbit Anti-Human Albumin</td>
			<td>Dako</td>
			<td>A0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene Sorbitan Monolaurate (Tween 20)</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>28353-85</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>189-02025</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium L-Ascorbate</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>03422-32</td>
			<td></td>
		</tr>
		<tr>
			<td>StemFit AK02N</td>
			<td>REPROCELL</td>
			<td>RCAK02N</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS (10x)</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>12748-31</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine, methyl ester (TMRM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>T668</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>28229-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>20577-34</td>
			<td></td>
		</tr>
		<tr>
			<td>TRYPSIN 250</td>
			<td>Difco</td>
			<td>215240</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptose phosphate broth solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8159</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of live mitochondria staining in cell-sorting to purify hepatocytes derived from human induced pluripotent stem cells

인간 배아 줄기(ES) 및 유도 만능 줄기(iPS) 세포는 무제한 증식 및 만능 특성으로 인해 심각하게 병든 장기를 치료하기 위한 세포 기반 재생 의학에 잠재적으로 응용될 수 있습니다. 그러나 인간 ES/iPS 세포를 100% 순수 표적 세포 유형으로 분화시키는 것은 환경에 대한 민감도가 높기 때문에 어렵습니다. 이식 후 종양 형성은 오염, 증식, 미분화 세포에 의해 발생하므로 재생 의학의 안전한 실현을 위해서는 고순도 기술이 필수적입니다. 종양 형성의 위험을 완화하기 위해 인간 iPS 세포 유래 간세포에 대한 고순도 기술이 개발되었습니다. 이 방법은 유전자 변형 없이 높은 미토콘드리아 함량과 세포 표면 마커 ALCAM(활성 백혈구 세포 접착 분자)의 조합을 사용하여 FACS(형광 활성화 세포 분류)를 사용합니다. 이 방법을 사용하여 정제된 간세포의 97% ± 0.38%(n=5)에서 알부민 단백질 발현을 나타냈다. 이 기사는 인간 iPS 세포를 간세포로 분화하는 가장 최근의 2차원 분화 방법에 적용되는 이 방법에 대한 자세한 절차를 제공하는 것을 목표로 합니다.

Embryonic and induced pluripotent stem cells (ES and iPS, respectively) are considered promising cell sources for regenerative therapies. However, the efficiency of differentiating these cells into specific target cell types can vary, even when using the same cell line, protocol, and experimenter1,2,3,4. This variability may be attributed to the high sensitivity of human ES/iPS cells to their environment. Therefore, it is currently difficult to consistently obtain pure target cells. To achieve highly safe regenerative medicine, it is crucial to eliminate proliferative cells and undifferentiated stem cells in therapeutic cells, and advanced purification technology for target cells is essential5,6,7.
A cell sorter is a device that instantaneously analyzes individual cells and sorts live cells of interest based on the fluorescent signal strengths, offering a promising solution. This can be accomplished through antibody staining of cell type-specific surface markers or by utilizing cell type-specific reporter gene expressions. Using this technique, there are several reports on methods for purifying pluripotent stem cell-derived cardiomyocytes8,9,10 and hepatocytes11,12. Hattori et al. developed an innovative mitochondrial purification method using a cell sorter13. Taking advantage of the fact that cardiomyocytes have high energy demands through mitochondrial activity, staining the cells with the live mitochondria-indicative dye TMRM (tetramethylrhodamine methyl ester) can be used to label and highly purify cardiomyocytes by FACS from human ES cell-derived embryoid bodies containing various cell types. The absence of tumorigenicity was confirmed by teratoma formation assays with the purified cardiomyocytes. Furthermore, Yamashita et al. unexpectedly discovered a method to purify hepatocytes from human ES cell-derived embryoid bodies by isolating fractions with high mitochondrial activity and ALCAM-positive expression14. The rationale for this method is that hepatocytes also have a relatively high number of mitochondria due to their high consumption of ATP for nutrient metabolism and detoxification15, and hepatocytes express ALCAM, a member of the immunoglobulin superfamily, which plays a role in cell adhesion and migration16.
Previous highly purifying methods for pluripotent stem cell-derived hepatocytes required genetic modifications, and non-genetic purification methods had low efficiency17. The mitochondrial non-genetic method holds merit for achieving high purity. When hepatic progenitor cells are needed, CD133 and CD13- or Dlk1-based methods18,19 can be chosen. Although the accuracy of genome editing technology has advanced, the potential risk of unforeseen genomic changes (e.g., carcinogenesis) cannot be entirely eliminated. Methods based on mitochondrial activity, without involving genetic modification, can be free from such risks.
As a result of examining various mitochondrial indicators in neonatal rat cardiomyocytes, TMRM was observed to disappear completely within 24 h, whereas other dyes remained for at least 5 days13. Moreover, it is important to note that TMRM and JC-1 did not impact cell viability when assessed with the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while other dyes demonstrated varying effects on cell viability13. TMRM demonstrates higher safety.
To date, several two-dimensional (2D) differentiation methods have been developed as more efficient approaches for inducing differentiation compared to three-dimensional (3D) embryoid body formation. This is because step-wise differentiation-inducing compounds or cytokines can be uniformly administered to cells on a 2D plane, rather than in a 3D space. In this study, the previously reported method20 was modified to induce differentiation into human iPS cell-derived hepatocytes. Here, the details of the procedures for the up-to-date 2D differentiation and purification of hepatocytes derived from human iPS cells are described.

저자 스포트라이트: 재생 의학을 위한 인간 유도 만능 줄기 세포의 간세포 정제 발전

인간 유도 만능 줄기 세포에서 유래한 간세포를 정제하기 위한 세포 분류에 살아있는 미토콘드리아 염색 적용

The scope of our research is to purify human induced pluripotent stem cell-derived hepatocytes, using without genetic modification. We aim to address the issue of the tumorigenic risk associated with the undifferentiated cells, among those differentiated from human iPS cells. One of the current experimental challenges is a low cell purity, due to the mechanical storage of sorting.We are developing a new purification method, that utilizes the unique metabolism features of hepatocytes, for large-scale purification. One non-cell sorting method for hepatic progenitor cells cannot obtain pure hepatocytes. Another one for mature hepatocytes can only treat a small fraction of hepatocytes, because of the limited expression of the marker protein.In contrast, our approach can obtain hepatocytes at different maturation levels with a higher eve. Human iPS cell-derived hepatocytes are extremely immature, and show more reduced hepatocyte macro expression after purification. Our future goal is to enhance hepatocyte modulation using organoidal technology and day-to-day advancements to regenerative medicine and drug discovery.

인간 iPS 세포가 2D 배양을 통해 간세포로 분화하도록 유도하는 과정(그림 1A)과 대표적인 세포 특징(그림 1B)의 타임라인이 표시됩니다. 대략 분화 12일째에 세포는 간세포의 특징인 다각형 세포 모양과 둥근 핵을 나타내기 시작했습니다. 일부 간세포는 또한 다핵형성(multinucleation)을 보였다.
FACS 분석은 분화 27일째에 세포를 사용하여...

Watch this Scientific Journal Video about Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells at JoVE.com

만능 줄기 세포에서 유래한 간세포는 미토콘드리아 및 활성 백혈구 세포 접착 분자(ALCAM, CD166이라고도 함) 염색을 사용하여 세포 분류를 통해 정제할 수 있습니다.

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely ...

본 연구의 범위는 유전자 변형 없이 인간 유도 만능줄기세포 유래 간세포를 정제하는 것입니다. 우리는 인간 iPS 세포와 분화된 세포 중 미분화 세포와 관련된 종양 유발 위험 문제를 해결하는 것을 목표로 합니다. 현재 실험적 과제 중 하나는 선별의 기계적 저장으로 인한 낮은 셀 순도입니다.간세포의 독특한 대사 특성을 활용하여 대규모 정제를 위한 새로운 정제 방법을 개발하고 있습니다. 간 전구 세포에 대한 한 가지 비세포 분류 방법으로는 순수한 간세포를 얻을 수 없습니다. 성숙한 간세포를 위한 또 다른 방법은 마커 단백질의 발현이 제한되어 있기 때문에 간세포의 극히 일부만 치료할 수 있습니다.대조적으로, 우리의 접근 방식은 더 높은 이브와 함께 다른 성숙 수준에서 간세포를 얻을 수 있습니다. 인간 iPS 세포 유래 간세포는 매우 미성숙하며 정제 후 간세포 매크로 발현이 더 감소합니다. 우리의 미래 목표는 유기 세포 기술을 사용하여 간세포 조절을 강화하고 재생 의학 및 약물 발견에 대한 일상적인 발전을 향상시키는 것입니다.

application-live-mitochondria-staining-cell-sorting-to-purify

Research

JoVE Journal

Biology

Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely diseased organs due to their unlimited proliferation and pluripotent properties. However, differentiating human ES/iPS cells into 100% pure target cell types is challenging due to their high sensitivity to the environment. Tumorigenesis after transplantation is caused by contaminated, proliferating, and undifferentiated cells, making high-purification technology essential for the safe realization of regenerative medicine. To mitigate the risk of tumorigenesis, a high-purification technology has been developed for human iPS cell-derived hepatocytes. The method employs FACS (fluorescence-activated cell sorting) using a combination of high mitochondrial content and the cell-surface marker ALCAM (activated leukocyte cell adhesion molecule) without genetic modification. 97% &plusmn; 0.38% (n = 5) of the purified hepatocytes using this method exhibited albumin protein expression. This article aims to provide detailed procedures for this method, as applied to the most current two-dimensional differentiation method for human iPS cells into hepatocytes.

Hepatocytes derived from pluripotent stem cells can be purified through cell sorting, using a combination of mitochondrial and activated leukocyte cell adhesion molecule (ALCAM, also known as CD166) staining.

연구

생물학

Hepatic Differentiation of Human-Induced Pluripotent Stem Cells in 2D Cultures

To begin, coat each well of a six-well plate with one milliliter of basement membrane matrix and incubate at 37 degrees Celsius for one hour. Remove the coated matrix from the plate and add human-induced pluripotent stem cells prepared in AK02N medium supplemented with 10 micromolar ROCK inhibitor. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for three days.After three days, on day zero of the initiating hepatic differentiation, wash the cells once with RPMI 1640 medium. Next, to initiate endoderm differentiation, add RPMI B27 GlutaMAX supplemented with three to six micromolar CHIR 99021 and 100 nanograms per milliliter Activin A into the plate. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours.On day one, replace the existing medium with RPMI B27 GlutaMAX supplemented with 50 nanograms per milliliter of Activin A.Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours. On day two, to induce hepatic progenitor differentiation, replace the existing medium with RPMI B27 GlutaMAX medium supplemented with 1%dimethyl sulfoxide, and continue incubation for up to five days. On day seven, to initiate hepatocyte maturation, switch to hepatocyte maturation medium and incubate for 20 days at 37 degrees Celsius and 5%carbon dioxide.On day 27, human-induced pluripotent stem cells displayed polygonal cell shapes and rounded nuclei, characteristics of hepatocytes.

2D 배양에서 인간 유도 만능 줄기 세포의 간 분화(Hepatic Differentiation of Human-Induced Pluripotent Stem Cells)

Cell Preparation for Purification of Human-Induced Pluripotent Stem Cells-Derived Hepatocytes

After 27 days of initiating hepatic differentiation of human induced pluripotent stem cells, wash the cells with two milliliters of PBS per well. Prepare the enzyme solution in ADS buffer using the given components. Add one milliliter of solution per well of a six well plate.Place the plate at 37 degrees Celsius on a rotating shaker for about two hours to detach the cells from the plate. Confirm the cell detachment under a microscope, then pipette the cell suspension to disperse into single cells, and collect them in a 50 milliliter tube containing the hepatocyte maturation medium. Centrifuge the cell suspension at 200G for five minutes at 18 degrees Celsius.And using an aspirator, remove the supernatant. To stain the mitochondria of cells, resuspend the pellet in five milliliters of hepatocyte maturation medium containing 100 nanomolar TMRM. Incubate the cells for 30 minutes at 37 degrees Celsius while protecting them from light.Again, centrifuge the cell suspension and resuspend the pellet in one to five milliliters of cold ADS buffer containing 2%FBS. After cell counting, aliquot 500, 000 cells to a 1.5 milliliter tube and label it as the no primary antibody control. Centrifuge the remaining cell suspension at 200G for five minutes at four degrees Celsius, and remove the supernatant.Add 100 microliters of diluted primary antibody per one million cells. Transfer the cell suspension to a 1.5 milliliter tube and incubate on ice for 50 minutes. After incubation, centrifuge the cell suspension and wash the cells twice with cold ADS buffer containing 2%FBS.Now add 100 microliters of diluted secondary antibody per one million cells to the primary antibody, stained or unstained cells, and incubate for 30 minutes on ice. Again, centrifuge the cell suspension and wash the cells twice with cold ADS buffer containing 2%FBS. After resuspending the cells in ADS buffer, filter them through a snap cap cell strainer and place the tube on ice.

인간 유도 만능 줄기 세포 유래 간세포의 정제를 위한 세포 준비

Fluorescence-Activated Cell Sorting of Hepatocytes Derived from Human Induced Pluripotent Stem Cells

To begin, set up the fluorescence-activated cell sorting, or FACS machine, for cell sorting following the manufacturer's instructions. Place the sample tube on the FACS machine and load it. Gate TMRM-high and ALCAM negative population in the P1 region to sort non-hepatocytes.Then gate the TMRM high and ALCAM positive population in the P2 region to sort hepatocytes. Collect sorted cells in 15-milliliter collection tubes containing hepatocyte maturation medium. After sorting, remove the collection tube from the FACS machine.Centrifuge the sorted cell suspension at 200 G for five minutes at 18 degrees Celsius. Remove the supernatant, and re-suspend the pellet in two milliliters of hepatocyte maturation medium with 10 micromolar rock inhibitor and 20 nanomolar cyclosporine A.Seed the cells on mitomycin C-treated mouse embryonic fibroblasts with hepatocyte maturation medium in a six-well plate. Culture the cells in a 37-degree Celsius carbon dioxide incubator for five days before immuno staining for hepatocyte purity test.FACS analysis conducted on day 27 of hepatic differentiation revealed TMRM high and ALCAM positive population. The immunohistochemical staining of P1 and P2 cells cultured on mouse embryonic fibroblasts after sorting is shown. A purity test by counting albumin-positive cells showed 0%of P1, and approximately 97%of P2 cells were hepatocyte-like cells.