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기사 소개

  • 요약
  • 초록
  • 서문
  • 프로토콜
  • 결과
  • 토론
  • 공개
  • 감사의 말
  • 자료
  • 참고문헌
  • 재인쇄 및 허가

요약

여기, 우리는 기능, 계약, cardiomyocyte 같은 세포를 생성 하기 위해서는 인간 중간 엽 줄기 세포의 젊은 소스의 심장 감 별 법 잠재력을 효율적으로 활용 하는 방법을 제시 생체 외에서.

초록

심근 경색과 이후 허 혈 성 캐스케이드 congestive 심장 마비, 전세계 사망률의 주요 원인으로 이어지는 cardiomyocytes의 광범위 한 손실 될. 중간 엽 줄기 세포 (MSCs)는 현재, 침략 적 기술을 대체 하 세포 기반 치료를 위한 유망한 옵션입니다. MSCs 엽 계보, 심장 세포 유형, 등으로 분화 할 수 있다 하지만 기능 세포로 완전 한 차별화가 아직 달성 하지. 차별화의 이전 방법 약리학 대리인 또는 성장 요인에 근거 했다. 그러나, 더 순수 관련 전략 cardiomyogenic 변환을 MSCs를 사용할 수도 있습니다. 여기, 우리가 cardiomyocyte 같은 계약 셀 생산 cardiomyocyte 급지대 레이어에 MSC 집계를 사용 하 여 차별화 방법 제시.

인간 탯 줄 혈관 주위 세포 (HUCPVCs) 보다 잠재적인 큰 차별화를가지고 표시 되었습니다 일반적으로 MSC 종류, 골 MSCs (BMSCs) 등을 조사. Ontogenetically 젊은 소스로 서, 우리는 이전 소스에 비해 첫 삼분기 (FTM) HUCPVCs의 cardiomyogenic 잠재력 조사. FTM HUCPVCs는 그들의 utero immunoprivileged 속성 경작 하는 때 유지 하는 MSCs의 소설, 풍부한 소스 생체 외에서. 이 분화 프로토콜, FTM 및 용어를 사용 하 여 (즉, myocyte 증강 2 C, 심장 분 T, 무거운 체인 심장 myosin, 신호 규제 단백질 α, 비율과 connexin 43) cardiomyocyte 마커의 증가 식에 표시 된 대로 HUCPVCs BMSCs에 비해 크게 증가 cardiomyogenic 차별화를 달성. 그들은 또한 그들의 더 낮은 HLA A 식 및 높은 HLA-G 식 시연으로 상당히 낮은 immunogenicity를 유지. 잠재력과 셀 클러스터 심장 급지대 레이어에 공동 문화의 1 주 안에 계약, 그렇게 할 첫 번째 MSC 종류 되 고 생성 된 집계 기반 차별화 적용, FTM HUCPVCs 증가 집계 형성을 보였다.

우리의 결과이 차별화 전략 효과적으로 FTM HUCPVCs 같은 젊은 MSCs의 cardiomyogenic 잠재력을 활용 하는 체 외에서 사전 차별화 그들의 재생 효능 비보를 증가 하는 잠재적인 전략 될 수 보여줍니다.

서문

심부 전 (CHF) 병 적 상태와 사망률 전세계의 주요 원인으로 지속합니다. CHF는 종종 cardiomyocytes의 대규모 손실과 심근 경색 (MI)1의 병 적인 결과로 흉터 조직 세포 무료의 개발에 따라 발생 합니다. 마음은 부분적으로 자체 갱신 기관, 풍요로 움과 노인된 환자에서 종종 손상 후 복구에 대 한 부족 한 되는 기능에 거주 줄기와 조상 세포 수영장 크게 조직 재생을 실행에 대 한 책임 감소 한다. 따라서, 손상 된 심근으로 건강 한 기증자 세포의 이식 관련 된 실험적 치료 개발에 큰 관심이입니다. 그것은 기증자 세포 조직, 구조를 복원 뿐만 아니라 또한 영향을 받는 심근의 기능 회복을 달성.

기본 심장 심장 조직-거주자를 고용 하 고 생 골 수 유래 줄기 세포 후 부상에 대 한 복구2,,34. 재생 세포 호스트 및 기증자 파생 모두 해야 합니다 적절 한 표현 형 및 효율적이 고 안전 하 게 손실 된 세포를 대체 하는 능력과 함께 개장 심근의 microenvironment에서 기능 능력을가지고. 차별화 방법 체 외에서 높은 효율, 줄기 세포 기반 cardiomyocyte 생산5,6을 달성 하기 위해 광범위 하 게 사용 되었습니다. 심장 혈통 마커의 식 프로필 심장 계보7으로 줄기 세포 분화 과정을 정의 하는 데 사용 됩니다. 초기 분화 마커, NKX2.5, myocyte 증강 인자 2 C 등 (Mef2c), 및 GATA48,9, cardiomyogenic 프로세스의 개시의 표시 될 수 있습니다. 성숙한 cardiomyocyte 마커 차별화 효능을 평가 하기 위해 일반적으로 사용 되 있으며 신호 규제 단백질 α (SIRPA)10, 심장 분 T (cTnT)11, 무거운 체인 심장 myosin (MYH6)8,,1213, connexin 43 (Cx43)14,,1516. 배아 줄기 세포 (ESCs)와 만능 줄기 세포 (Psc)를 사용 하 여 방법 철저 하 게 최적화 하 고 되었습니다 유도 요인, 산소 및 영양소 그라디언트, 세부 사항 및 작업5,6,7,,1718의 정확한 타이밍에 관한 논의. 그럼에도 불구 하 고, esc 키 및 PSC 기반 기술을 여전히 여러 윤리 및 안전 우려, 차선 electrophysiological 및 면역 기능19,20함께 제시. 종종 이러한 세포와 함께 이식 하는 호스트 immunorejection 경험과 영구 면역 억제 필요. 이것은 주로 중요 한 조직 적합성 복잡 한 (MHC) 분자 호스트에 기증자 및 결과 T 세포 응답21일치. 동안 개별 MHC 종류 I 정합은 가능한 해결책, 더 접근 가능한 임상 연습 immunoprivileged 거절의 우려를 극복 하기 위해 보편적으로 셀 소스를 요구할 것입니다.

특히, BMSCs, 임상 응용, MSCs에서에서 사용에 대 한 대체 셀 소스로 199522에 그들의 초기 설명 이후 조직 재생에 사용 하기 위해 조사 했습니다. MSCs 거주 재생 세포 거의 모든 vascularized 조직23에서 찾을 수 있는 것으로 추정 된다. 원하는 소스에서 격리, 따라 MSCs 문화에 쉽게 확장 될 수 있다, 광범위 한 paracrine 용량 있고 종종 소유 immunoprivileged 또는 immunomodulatory 속성24,25. 그들의 안전과 효능 이미 표시 되었습니다 여러 전 임상 연구에서 특히 심장 재생3,26.

많은 MSC 차별화 전략 변수 효율 5 azacytidine22 와 DMSO27그리고 성장 또는 Bmp5,7,,2829 또는 angiotensin II30, 같은 morphogenic 요인 등의 약리 에이전트를 사용합니다. 그러나 이러한 전략, 기반 하지 순진한 재생 셀은 유도 또는 상해의 위치에 배달 되 고 후 발생할 가능성이 장애물 vivo에서. 더 순수 관련 전략, 더 어려운 동안 정의 및 조작, MSC 차별화 자체 조직 microenvironment에서 신호를 통해 유도 될 수 있다 전제에 기반. 이전 연구 보여주었다 심장 세포 lysates31 또는 심 실 myocardium32,33에 노출 또는 기본 cardiomyocytes 체 외 에서15,34, 접촉 MSCs에서 심장 감 적의 식을 높일 수 있습니다 직접. Cardiomyocytes39,40 BMSCs 융합 초기 심근을 생성 하는 경우, 비록 다른 MSCs35,36,,3738와 심장 부상 치료 후 자연 스러운 cardiomyogenesis를 증명 하고있다. 우리의 지식, 기능, 자발적 계약 cardiomyocytes 조직 소스 (hMSCs) 인간의 MSCs에서 하지 아직 보고 되었습니다.

현재 일치는 모든 MSCs 혈관 주위 세포23에서 발생 하는 것 이다. 영 MSCs pericyte 속성 인간 탯 조직41,,4243의 혈관 주위 지역에서 격리 될 수 있습니다. BMSCs에 비해 HUCPVCs 소유 증가 감 별 법 잠재력 및 다른 여러 재생 장점, 두 생체 외에서41,44 그리고 vivo에서45,46,47. 특히, 산 모-신생아 인터페이스 되는 소스 HUCPVCs는 MSCs의 성인 소스에 비해 상당히 낮은 immunogenicity. 우리의 연구에 초점을 맞추고 특성화 및 FTM HUCPVCs, 조사, MSCs의 막내 소스의 전 임상 응용 프로그램 우리 이전 증식 및 높은 multilineage 증가 표시는 차별화 능력, cardiomyogenic 계보41에 포함 한.

여기, 선물이 MSCs 집계의 완전 한 cardiomyogenic 차별화를 달성 하기 위해 유도 세력 제공 조건에 vivo에서 2D 부착 문화에 비해 더 나은 모델 3D 환경으로 집계 형성과 기본 심장 셀 피더 레이어를 결합 하는 프로토콜. 심장 급지대 레이어를 활용 하 여 MSCs에 대 한 궁극적인 이식 사이트의 대표 하는 환경을 제공 합니다. 사전 또는 출생 탯에서 격리 하는 MSCs의 젊은 소스 형태로 집계를 유지 하면서도 그들의 면역 권한 성인 BMSCs에 비해 심장 형에 도달 하는 높은 능력을가지고 설명 합니다. 심장 혈통 마커 유전자와 세포의 유도 식의 가파른 상승 외 (즉, cTnT와 MYH6) 및 세포 표면 단백질 (즉, SIRPA 및 Cx43) cardiomyocytes에 대 한 특정, 우리 보여 FTM HUCPVCs의 감 별 법 잠재력이이 방법으로 무력화 될 수 있습니다 그리고 그들은 저절로 cardiomyocyte 같은 셀 계약에 상승을 줄 수 있습니다.

프로토콜

All studies involving animals were conducted and reported according to ARRIVE guidelines48. All studies were performed with institutional research ethics board approval (REB number 454-2011, Sunnybrook Research Institute; REB 29889, University of Toronto, Toronto, Canada). All animal procedures were approved by the Animal Care Committee of the University Health Network (Toronto, Canada), and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, 8th edition (National Institutes of Health 2011).

1. Tissue Culture

  1. Culture FTM HUCPVCs, term HUCPVCs (previously established, n ≥ 3 independent lines for each)42 and commercially available BMSCs in alpha-minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and a 1% penicillin/streptomycin (P/S) cocktail. Culture rat primary cardiomyocytes and MSC-cardiomyocyte co-cultures in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM-F12) containing 10% FBS and 1% P/S.
    NOTE: Sterilize the medium using a 0.2-µm filter. Store prepared medium solutions at 4 °C for up to 3 weeks.
  2. Maintain cell cultures in humidified incubators (95% relative humidity, 37 °C, and 5% CO2) and passage at 70-80% confluency, determined by phase-contrast microscopy. Use appropriate volumes of medium for the size of tissue culture dish used (e.g., 10 mL in a 10-cm dish and 2 mL per well in 6-well tissue culture plate). Use these culture conditions for the duration of the protocol.
  3. Dissociate MSC monolayers for passaging or MSC-cardiomyocyte co-culture establishment using a dissociation enzyme solution (2 mL/well in a 6-well plate) and incubate at 37 °C for 4 min.
  4. Transfer the dissociated cells to a 15-mL tube and centrifuge at 400 x g for 5 min.
  5. Aspirate the supernatant without disrupting the cell pellet and resuspend the cells in 1 mL of a culture medium appropriate for counting using an automated cell counter. Seed the cells as described in the following protocol sections.

2. Preparation of Primary Rat Cardiomyocyte-MSC Co-cultures

  1. Obtain heart tissue for primary cardiomyocyte isolation.
    1. Euthanize rat pups (5-6 days postnatal) using CO2 asphyxiation. Set CO2 chambers to 20% gas replacement (flow rate = 0.2 x chamber volume per min). Confirm exitus by the absence of the pinch reflex.
    2. Remove the atria with the connecting major blood vessels using sterilized instruments (i.e., forceps and curved scissors)41. Transfer the hearts to 50-mL tubes containing sterile PBS with1% P/S (PBS-P/S) on ice.
    3. Cut the ventriculi in half and let the blood wash out in a 10-cm dish with 10 mL of PBS-P/S on ice. Cut the ventricular walls into small pieces (diameter = 2-3 mm) using curved scissors.
    4. Transfer the heart pieces from 10-12 animals to a 50-mL tube using a serological pipette and let them settle.
    5. Remove as much PBS-P/S as possible without removing any heart pieces. Add 10 mL of new PBS-P/S.
  2. Digest the heart tissue to isolate the cardiomyocytes.
    1. Allow the heart pieces to settle. Replace the PBS-P/S with 10 mL of 0.15% trypsin in PBS and shake at 37 °C for 10 min.
    2. Discard the supernatant. Repeat the digestion described in step 2.2.1 three more times, but decant the supernatants into 50-mL collection tubes containing 10 mL of 100% FBS.
  3. Centrifuge the cells (400 x g, 5 min) and aspirate the supernatant. Resuspend the cells in DMEM-F12 containing 10% FBS and 1% P/S and seed onto a 6-well plate (1 x 105 cells/cm2, 2 mL of medium per well).
  4. After 1 h, transfer the medium containing non-attached cells to a 50-mL tube and discard the attached cells. Count the cells in suspension and re-plate them into new 6-well plates (1 x 105 cells/cm2, 2 mL of DMEM-F12 containing 10% FBS and 1% P/S per well).
  5. Inhibit cell proliferation with bromodeoxyuridine (BrdU).
    Caution: BrdU is a strong teratogen and suspected mutagen. Please ensure proper training is provided and refer to the safety data sheet before use.
    1. Once cells have attached, replace the medium in the 6-well plate with DMEM-F12 containing 10% FBS, 1% P/S (2 mL of medium per well), and 5 µM BrdU. Incubate for 16 h (37 °C, 5% CO2).
    2. Remove the BrdU-containing medium and replace with DMEM-F12 containing 10% FBS and 1% P/S (2 mL of medium per well).
  6. Prepare pre-stained MSCs.
    1. Once MSC cultures are at 70-80% confluency in 10-cm dishes, remove the culture medium and add 3 mL of cell dissociation solution. Incubate the dish at 37 °C and 5% CO2 for 5 min.
    2. Transfer the dissociated cells to a 15-mL tube and centrifuge at 400 x g for 5 min.
    3. Aspirate the supernatant without disrupting the cell pellet and resuspend the cells in 1 mL of DMEM-F12 containing 10% FBS and 1% P/S for counting using an automated cell counter.
    4. Dilute the cells to a concentration of 1 x 106 MSC/mL of DMEM-F12 containing 10% FBS and 1% P/S.
    5. Incubate the MSCs with viable, non-transferable fluorescent dye (5 µM, 30 min, 37 °C, 5% CO2) in 1.5-mL centrifuge tubes for 1 h.
    6. Centrifuge the tubes at 400 x g for 5 min. Aspirate the supernatant and resuspend the pellet in DMEM-F12 containing 10% FBS and 1% P/S for a cell concentration of 1 x 106 MSC/mL. Repeat this a total of 3 times.
  7. Transfer the MSCs onto cardiomyocytes (step 2.5.2) at a concentration of 10 x 104 cells per well of the 6-well plate.

3. Preparation of Aggregate Co-cultures

  1. Prepare a single-cell suspension of MSCs (2 x 104 cells/mL of medium, passage # ≤ 6) in alpha-MEM supplemented with 10% FBS and 1% P/S (see step 2.6).
    NOTE: Refer to section 1 of the protocol for the passaging of cells. Alternatively, pre-stain MSCs as per step 2.6.
  2. Initiate aggregate formation by placing 25-µL drops of cell suspension (500 cells) on the inner surface of the lids of 10-cm tissue culture dishes (up to 50 drops per lid). Place the lids on their bottom counterparts containing PBS-P/S. Incubate at 37 °C and 5% CO2.
    NOTE: Place 5-7 mL of PBS-P/S into the culture dish below the hanging drops to avoid drop evaporation.
  3. Observe aggregate formation in the drops after 3 days using a stereomicroscope. If over 40 out of 50 drops contain formed aggregates, collect the drops from the lids using a 1-mL micropipette and transfer the aggregates directly onto primary rat cardiomyocyte monolayers (prepared in steps 2.1-2.7; 10 drops/well). Avoid vigorous pipetting to preserve aggregate integrity.
  4. Keep aggregate co-cultures in the incubators for up to 2 weeks, changing the full volume of medium (2 mL of DMEM-F12 containing 10% FBS and 1% P/S per well) every 72 h.
    1. Daily observe aggregates attaching on feeder cell layers using bright-field microscopy. Record contracting aggregates when observed.
  5. Prepare aggregates for analysis.
    1. Remove the medium and add 2 mL of PBS per well of a 6-well tissue culture dish. Remove the PBS and add 2 mL of dissociation solution per well. Incubate for 3 min at 37 °C and 5% CO2.
    2. Centrifuge at 400 x g for 5 min to obtain a cell pellet. Resuspend in medium, as specified for the applications described in the subsequent steps (see steps 4.1, 5.1, and 6.1) and pass through a 70-µm cell strainer.

4. Flow Cytometry (FC) and Fluorescence-activated Cell Sorting (FACS)

  1. Incubate cell suspensions (1 x 105 cells in 200 µL of PBS containing 3% FBS) with fluorophore-conjugated (FITC or APC) primary antibodies (i.e., CD49f, Cx43, TRA-1-85, HLA-A, HLA-G, and SIRPA for FC or TRA-1-85 for FACS; 1:40) at 4 °C for 30 min, protected from light.
  2. Centrifuge (400 x g, 5 min) and resuspend the cells in 1 mL of PBS with 3% FBS for FC or PBS with 0.5% FBS for FACS.
    NOTE: The FC of MSCs was optimized by Hong et al.41.
  3. Maintain the cells at 4 °C in the dark until they are ready to be analyzed by FC (at least 1 x 104 events) or FACS. Sort the cells as described41. Re-plate TRA-1-85 high-positive sorted cells in 6-well plates (1 x 104 cells/well, 2 mL of DMEM-F12 containing 10% FBS and 1% P/S) within 1 h.
    NOTE: For the gating strategy of the TRA-1-85 human cell surface antigen, see the Supplementary Figure.

5. Immunocytochemistry (ICC) and Microscopy

  1. Re-plate the cell suspensions obtained from the co-cultures (step 3.5.2) or FACS (section 4) onto chamber slides (1 x 104 cells/well, 2 mL of DMEM-F12 containing 10% FBS and 1% P/S per well). Let the cells attach overnight in a tissue culture incubator (see section 1 for the conditions).
  2. Fix the cells using 3 mL of 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature. Wash 3 times with 3 mL of PBS containing 1% bovine serum albumin (BSA; PBS-BSA) for 5 min per wash.
    Caution: Wear appropriate personal protective equipment when handling PFA.
  3. Permeabilize the cells in 3 mL of PBS-BSA with 0.1% Triton X-100. Incubate at room temperature for 10 min for intracellular antigens (i.e., alpha sarcomeric actinin (aSarc) and Cx43), or 25 min for intra-nuclear antigens (i.e., Mef2c and human nuclear antigen (HuNu)). Wash 3 times with 3 mL of PBS-BSA for 5 min per wash.
  4. Block the samples against non-specific antibody reactions with 3 mL of PBS containing 5% normal goat serum (NGS) and 1% BSA for 15 min at room temperature. Wash 3 times with 3 mL of PBS-BSA for 5 min per wash.
  5. Incubate the cells in the primary antibodies (i.e., Mef2c, aSarc, Cx43, and HuNu) diluted 1:200 in 3 mL of PBS-BSA at 4 °C overnight.
  6. Wash 3 times with 3 mL of PBS-BSA for 5 min per wash and incubate with secondary antibodies for 30 min at room temperature. Wash 3 times with 3 mL of PBS-BSA for 5 min per wash.
  7. Store the stained specimens in 3 mL of of mounting medium.
  8. Acquire images using a fluorescence microscope. Use a 10X objective (NA = 0.3), and a 20X objective (NA = 0.45) for lower-magnification imaging. Use fluorescence filter cubes and wavelengths for GFP (ex = 470/22 nm, em = 525/50 nm) and RFP (ex = 531/40 nm, em = 593/40 nm) for the secondary antibodies used (see the Materials and Equipment Table).
  9. Quantify images using imaging software (see the Materials and Equipment Table for the recommended software). Normalize the fluorescence intensity readings to the secondary control acquisitions.

6. RNA Isolation and Quantitative RT-PCR

  1. Prepare RNA samples from undifferentiated MSC cultures or MSCs sorted from co-cultures using column-based RNA isolation, according to the manufacturer's instructions. Prepare 1 x 104 to 1 x 106 cells in 0.7 mL of cell lysis buffer (provided with the RNA isolation kit) per sample.
  2. Prepare cDNA from up to 2 µg of RNA per 100-µL RT reaction.
  3. Perform qPCR using 10 ng of cDNA per reaction (40 cycles, 60 °C annealing/extending temperature).
    1. Use primers for human MY6H and cTnT in a 500-nM concentration and 1-100 ng of cDNA per reaction (see the Materials and Equipment Table). Use GAPDH, ACTB, and HPRT as internal housekeeping normalizers. Use commercially available human-induced pluripotent stem cell-derived cardiomyocytes as a positive control.
      NOTE: Express the fold-change of expression compared to undifferentiated MSC-derived cDNA samples.

결과

HUCPVCs Display Higher Aggregate-formation Potential and CD49f Expression Levels Compared to BMSCs:

To induce the differentiation of hMSCs (i.e., FTM HUCPVCs, term HUCPVCs, and BMSCs), single-cell suspensions of undifferentiated MSCs or MSC-containing hanging drops (Table 1) were transferred onto rat primary cardiomyocyte monolayers to establish direct co-cultures or aggregate co-cultur...

토론

줄기 세포의 심근 분화 MSC 소스에서 cardiomyocyte 같은 세포를 생성 하는 데 사용 되 고 여러 가지 다른 전략으로 2 년간 개발 되었습니다. 그러나 이러한 전략은,, 많은, 고 사용 조건이 자주 하지는 환경 이식 세포 발생에서 vivo에서의 대표.

기존의 방법 달리 여기에 제시 된 프로토콜 기본 심장 급지대 레이어 및 MSC 집계 형성의 조합을 사용 합니다. 기본 심장 급지대 레이?...

공개

닥터 클 리 포드 L. Librach는 특허의 공동 소유자: 격리의 첫 번째 임신 탯 조직에서 파생 된 셀의 사용 방법, 캐나다와 호주에 부여.

감사의 말

저자는 다음 직원을 감사 하 고 그들의 공헌에 대 한 인사를 연구: 매튜 Librach, 라일라 Maghen, 타 냐 A. Baretto, Shlomit Kenigsberg, 그리고 앙드레 Gauthier 피셔. 이 작품은 온타리오 연구 기금-연구 우수성 (ORF-다시, 라운드 #7)과 만드는 프로그램 i n c.에 의해 지원 되었다

자료

NameCompanyCatalog NumberComments
0.25% Trypsin/EDTAGibco25200056For cell dissociation
Alpha-MEMGibco12571071For HUCPVC and BMSC culture media.
PE-conjugated anti-human/mouse CD49f antibodyBiolegend313612Integrin marker for FC
APC-conjugated human Cx43/GJA1 antibodyR&D SystemsFAB7737AConnexin 43 marker for FC
FITC-conjugated HLA-A2 antibodyGenway Biotech Inc.GWB-66FBD2Immunogenicity marker for FC
FITC-conjugated anti-HLA-G [MEM-G/9] antibodyAbcamab7904Immunogenicity marker for FC
FITC-conjugated mouse anti-human SIRPA/CD172a antibodyAbD Serotec/Bio-RadMCA2518FCardiac marker for FC
APC-conjugated human TRA-1-85/CD147 antibodyR&D SystemsFAB3195AHuman cell marker for FC and FACS
FITC-conjugated human TRA-1-85/CD147 antibodyR&D SystemsFAB3195FHuman cell marker for FC and FACS
Anti-connexin 43/GJA1 antibodyAbcamab11370Cx43. For ICC
Goat anti-rabbit IgG (H+L) cross-absorbed secondary antibody, Alexa Fluor 555Life TechnologiesA-21428For ICC
Anti-sarcomeric alpha actinin [EA-53] antibodyAbcamab9465aSARC. For ICC
Goat anti-mouse IgM heavy chain cross-absorbed secondary antibody, Alexa Fluor 555Life TechnologiesA-21426For ICC
Mef2C (D80C1) XP rabbit antibodyNew England BioLabs Ltd.5030SFor ICC
Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488Life TechnologiesA-21206For ICC
Anti-nuclei (HuNu) (clone 235-1) antibodyEMD MilliporeMAB1281For ICC
MZ9.5 StereomicroscopeLeicaFor imaging aggregates.
1.5 ml centrifuge microtubesAxygenMCT-150-CFor staining MSCs with fluorescent dye.
ImageJOpen source image processing software.
Aria II BDUHN SickKids FC Facility. For cell sorting.
Bone marrow mesechymal stromal cellsLonzaPT-2501BMSCs
Bovine serum albuminSigma-AldrichA7030-100GBSA. To prepare solutions for ICC
BrdUEMD MilliporeMAB3424Caution: BrdU is a strong teratogen and suspected mutagen. Please ensure proper training and refer to the SDS before use.
Canto IIBDUHN SickKids FC Facility. For flow cytometry.
cDNA EcoDry PremixClontech/Takara639570For preparation of cDNA for qPCR
CellTracker Green CMFDA DyeLife TechnologiesC7025Fluorescent imaging of cell cytoplasm
Countess automated cell counterInvitrogen Inc.C10227For cell counting
DMEM-F12Sigma-AldrichD6421For rat primary cardiomyocyte culture medium.
Dulbecco's Phosphate Buffered SalineGibco10010023D-PBS, without Ca2+, Mg2+
EVOSLife TechnologiesIn-house fluorescent microscope
FACSCaliburBDIn-house. For flow cytometry.
Fetal bovine serum (Hyclone)GE HealthcareSH3039603FBS. Component of cell culture medium.
IDT Prime Time qPCR probesIntegrated Data TechnologiesFAM fluorophorehttp://www.idtdna.com/pages/products/gene-expression/primetime-qpcr-assays-and-primers
Lab Vision PermaFluor Aqueous Mounting MediumThermoScientificTA-030-FMFor storage of cells to undergo ICC
LSR II BDUHN SickKids FC Facility. For flow cytometry.
MoFlo AstriosBeckman CoulterUHN SickKids FC Facility. For cell sorting.
Normal goat serumCell Signaling Technology5425SNGS. Used in blocking solution for ICC
Nunc Lab-Tek II Chamber Coverglass, 8-wellsThermo Scientific Nunc155409To prepare samples for ICC
OmniPur Triton X-100 SurfactantEMD Millipore9410-OPAs a component of permeabilizing solution when preparing cells for ICC
Paraformaldehyde, 16% Solution, EM GradeElectron Microscopy Sciences15710For fixing cells for ICC.
Penicillin/streptomycinGibco15140122Component of cell culture medium.
PrimersSigmaCustom Standard DNA Oligos, Desalted, 0.2 μmolCTnT_F: GGC AGC GGA AGA GGA TGC TGA A; CTnT_R: GAG GCA CCA AGT TGG GCA TGA ACG A; MYH6 F: GCA AAG TAC TGG ATG ACA CGC T; MYH6 R: GTC ATT GCT GAA ACC GAG AAT G
Quorum Spinning Disk ConfocalZeissSickKids Imaging Facility
ReproCardio hiPS cell derived cardiomyocytesReproCellRCD001NPositive control for qPCR
RNeasy mini kitQiagen74106To isolate RNA for qPCR
Rotor-Gene SYBR Green PCR KitQiagen204074For qPCR with master mix
RPMI 1640GibcoA1049101For MSC, monocyte coculture medium.
TaqMan qPCR primer assaysThermo Fisher Scientific4444556For qPCR
Trypan BlueLife TechnologiesT10282Staining of cells for viability and counting
TrypsinGibco272500108For cell dissociation
VolocityPerkin-ElmerVolocity 6.3Imaging software
0.2 μm pore filterThermo Fisher Scientific566-0020For sterilizing tissue culture media
HERAcell 150i CO2 IncubatorThermo Fisher Scientific51026410For incubating cells
Dulbecco's phosphate buffered salineSigma-AldrichD8537PBS. 1X, Without calcium chloride and magnesium chloride
ForcepsAlmedic7727-A10-704For handing rat heart. Can use any similar forceps.
ScissorsFine Science Tools14059-11For mincing rat heart. Curved scissors recommended.
50 mL tubeBD Falcon352070For collection during cardiomyocyte collection and general tissue culture procedures
15 mL tubeBD Falcon352096For general tissue culture procedures
6-well platesThermo Scientific NuncCA73520-906For tissue culture
10 cm tissue culture dishesCorning25382-428For aggregate formation
Axiovert 40C MicroscopeZeissFor bright-field imaging through out tissue culture and the rest of the protocol
70 μm cell strainerFisherbrand22363548To ensure a single cell suspension before flow cytometry or sorting
Triton X-100EMD Millipore9410-1LUsed in permeabilization solution for ICC
Hoechst 33342Thermo Fisher ScientificH1399Stain used during visualization of Cx43 localization

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