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참고문헌 2022.03.14
[지방 줄기세포, AD-MSC 임상 논문] 건선 Cell Transplantation, 2016

 

Autologous Adipose-Derived Mesenchymal Stromal Cells for the Treatment of Psoriasis Vulgaris and Psoriatic Arthritis: A Case Report

Cell transplantation, 2016, http://dx.doi.org/10.3727/096368916X691998

Miguel M. De Jesus, Jayson S. Santiago, Camille V. Trinidad, Melvin E. See, Kimberly R. Semon, Manuel O. Fernandez Jr., and Francisco S. Chung

 

  Abstract

Psoriasis is a chronic inflammatory dermatologic disease exhibiting a wide spectrum of clinical signs. Its cutaneous manifestation is characterized by hyper proliferation of basal keratinocytes, thickened and scaly epidermis, and recruitment of inflammatory cells to the skin (12). T-cell infiltrates precede epidermal changes (lesions) (14), and bone marrow transplantation from psoriatic donors has led to T-cell infiltrates preceding epidermal changes (lesions) and development of psoriasis (15). Immune system dysregulation is now considered a root cause of the disease, which leads to the inflammatory and hyper proliferative manifestations. Genetics, environmental triggers, and infections also influence its occurrence and severity (8).

The basic characteristics of psoriatic lesions (redness, thickness, and scaliness) provide a means of assessing the severity of psoriasis. The current gold standard for the assessment of extensive psoriasis is the psoriasis area and severity index (PASI) (6). The PASI is a measure of the average redness, thickness, and scaliness of lesions, accounting for skin area. Psoriasis patients may also develop arthritis, ranging from mild and nondestructive symptoms, to severe and rapidly degrading arthropathy. Psoriasis is correlated with other conditions such as uveitis and, possibly, reduced life expectancy due to adverse events associated with the drugs used in treatment (12).

We investigated the use of autologous mesenchymal stromal cells (MSCs) instead of HSCs to avoid the safety problems associated with immune-ablation and allogeneic transplantation. We also assessed whether MSCs could provide clinical benefit due to their well-documented immunomodulatory properties (11). While the mechanisms of MSC-mediated immunosuppression are still uncertain, some functions of MSCs have been established: they inhibit T-cell proliferation in vitro, inhibit natural killer (NK) cell activity in vitro, disrupt the differentiation of HSCs and monocytes in vitro, and decrease the antigen-presenting capacity of dendritic cells. One study established the synergistic roles of interferon-g (IFN-g) and tumor necrosis factor-a (TNF-a) in the ability of MSCs to induce a regulatory/immunosuppressive phenotype in Th17 cells (a subset of T cells significant in psoriasis), downregulate IL-17 production, and increase IL-10 production (7). MSCs are currently being evaluated in several clinical trials on immune disorders such as Crohn’s disease, multiple sclerosis, type 1 diabetes, autoimmune hepatitis, and graft-versus-host disease. One trial is registered at the NIH ClinicalTrials.gov database utilizing umbilical cordderived MSCs for psoriasis (3). We explored the utility of adipose-derived MSCs (ADSCs) for the following reasons: relative ease of sample acquisition, abundance of MSCs in fat, and the observation that dermal MSCs in psoriasis patients have atypical properties (higher growth rates, varying gene expression patterns, and lower free radical-scavenging activity) (13). The main objectives were to measure safety and note any indicators of potential clinical utility for these conditions

 

  Method

Cell Harvest

Patients underwent mini liposuction procedures under tumescent anesthesia (lidocaine). About 200 ml of subcutaneous abdominal fat was aspirated and transported to the Cellular Therapeutics Center. Patients were discharged after no evidence of adverse reactions was observed

 

  Culture of Mesenchymal Stromal Cells

Lipoaspirate was washed three times with phosphatebuffered saline (PBS; pH 7.2) to separate the bloody fraction from the lipid fraction. Afterward, the lipid fractions were pooled and digested using collagenase I, collagenase II, and thermolysin (Roche, Mannheim, Germany) in a shaking water bath (37°C) for 1.5 h. The cell fraction was isolated via centrifugation and plated in complete culture media: Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY, USA), 10% fetal bovine serum (filter sterilized using a 0.20-µm microfilter; Life Technologies), 2 mM L-glutamine (Life Technologies), 100 U/ml penicillin (Life Technologies), and 100 µg/ml streptomycin (Life Technologies). Cells were fed every 4–5 days and passaged as necessary (up to passage 3) via enzymatic digestion with trypsin/ ethylenediaminetetraacetic acid (EDTA) (Life Technologies). All culture activities were performed under aseptic conditions in an ISO 14644-1 cleanroom class V facility with stringent quality control standards, and all reagents were tested for sterility beforehand

 

  Patient A, Case CTC P 0021 00 13AT01.

A 58-year-old Filipino male weighing 52 kg was diagnosed with psoriasis since 1987 and arthritis since 2008. The patient suffered from generalized erythematous scaly patches and thin plaques sparing the central face and soles of both feet; nail pitting and onycholysis on all fingernails and toe nails; scales distributed on the upper extremities, lower extremities, chest, abdomen, and bulk; and difficulty ambulating, standing, and bathing. Over the course of the patient’s disease, he underwent the following treatment modalities: clobetasol ointment, methotrexate, prednisone, etanercept, celecoxib, and various herbal remedies. He did not experience durable remission of symptoms at the time of consultation for MSC therapy.

 

  Patient B, Case CTC P 0030 00 14AT01.

A 28-year-old Filipino female weighing 45 kg was periodically receiving methotrexate weekly for a period of 5 years (from the age of 16 to 21) for PV. She was later diagnosed with hyperthyroidism at the age of 25. An attempt to wean off the steroid was instigated at age 22, but pulse doses were still required every month when symptoms flared. The patient also reported a variable therapeutic outcome using methotrexate. MSC therapy was considered with the goal of reducing the patient’s methotrexate dependence for her condition.

 

  Result

Patient A received two infusions of MSCs (days 0 and 40), and patient B received three infusions (days 0, 30, and 71). The timing of infusions was based on our unpublished study set by the attending physician. Both transplantation procedures were well tolerated, with no adverse reactions being recorded throughout the first year of follow-up. Patient A On the day of the first infusion, the PASI score of patient A was 21.6 (summarized in Fig. 1), with massive scaling, notable right knee swelling and pain, shoulder pain, finger pain, and toe pain. Forty days later, his PASI was 8.9, with notable decreases in erythema, induration, scaling, and lesions. Joint pain, condition of nails, and knee swelling were unchanged, although itching decreased significantly. The reduced PASI was sustained up to day 157, with noted reduction in onycholysis.

Photographic documentation for this patient is shown in Figure 2. The PASI of patient B during the time of consultation was 9.9, while receiving methotrexate, clobetasol, loratadine, and hydroxyzine. In order to prevent any untoward incident related to MSC–drug interactions, the patient was weaned off methotrexate for 23 days before the day of the first infusion. On the day of the first infusion, her PASI was 24 in the absence of medication such as clobetasol, anthralin, and hydroxyzine. On the day of the second infusion (30 days after the first), her PASI was 18.3 (summarized in Fig. 1); onycholysis on fingernails and toenails was noted. At this point, the patient was receiving topical clobetasol propionate, aquaphor, and betamethasone valerate. During the third infusion, her PASI reduced further to 9.4 with no indications of onycholysis or pitting. At this point, the patient was receiving topical clobetasol propionate, aquaphor, betamethasone valerate, and oral hydroxyzine medications. Forty-five days after, her PASI score was maintained at 9.6 (similar to levels with methotrexate use), with recurrence of onycholysis and pitting By the 231st day, her PASI was 8.3 while receiving calcipotriol, halobetasol–aquaphor, carbimazole, and antihistamines as needed. Onycholysis and pitting were sustained. The patient required methotrexate for the treatment of a recurrent flare after 292 days after MSC therapy.

 

>> 국문 번역 <<

 

  국문 제목

Autologous Adipose-Derived Mesenchymal Stromal Cells for the Treatment of Psoriasis Vulgaris and Psoriatic Arthritis: A Case Report

자가 지방 유래 중간엽 줄기세포를 통한 건선과 건선 관절염 치료 임상 사례 보고

 

  초록

건선은 다양한 증상을 동반하는 만성 염증성 질환이다. 그 증상으로는 각질 형성 세포 (Keratinocyte) 의 과 활성, 두껍고, 피부에 딱딱한 각질이 많이 일어남, 피부에 염증 반응 관여 세포가 많이 관찰됨. 한편, 건선 증상이 발현 하기 전에 대량의 T 세포가 그 주변으로 이동하는 것이 관찰되었으며, 건선 환자로부터 골수를 이식 받은 경우, 골수 수여자의 T 세포가 표피층으로 다량 이동하는 것을 확인하였다. 이처럼, 건선은 면역 체계 불균형에 의해 과다한 염증 반응 및 면역 세포의 과 활성에 의한 질병이다. 유전적 요인, 환경적 요인, 감염 등이 이를 심화시킬 수 있다.

환자의 증상을 통해 병의 중증도를 파악할 수 있다. 이를 표준화한 지표로는 Psoriasis Area and Severity Index (PASI) 가 있다. 일부 환자에게서 건선이 관절염을 유발하기도 하는데, 무증상의 느린 진행을 보이는 환자부터 극심한 통증을 동반하는 빠른 진행을 보이는 환자도 있다. 일부 환자는 포도막염을 동반하기도 하며, 치료를 위해 사용하는 의약품에 의한 심각한 부작용을 겪는 사례도 있다.

본 연구진은 조혈 줄기세포 이식을 통해 나타나는 면역 결핍 등의 문제를 피하기 위해 자가 중간엽 줄기세포를 사용해 면역 반응 조절에 관한 치료의 효용성을 평가하였다. 일부 연구를 통해 중간엽 줄기세포의 일부 면역 반응 관여 기전이 규명되었지만 (체외 실험 환경 내 T 세포의 활성 조절 확인, 체외 실험 환경 내 NK 세포의 활성 조절 확인, 체외 실험 환경 내 조혈 줄기세포와 단핵구 분화 억제 확인, 체외 실험 환경 내 수지상세포의 항원 제시 기능 억제 등), 중간엽 줄기세포가 체 내 에서 T 세포의 활성을 조절하는지는 여전히 확실치 않다. 한편, IFN-γ TNF-α 의 상호작용을 통해 중간엽 줄기세포가 Th17 을 통해 면역 억제 작용을 나타내고, IL-17 단백질의 발현은 억제시킴과 동시에 IL-10 은 촉진한다는 연구도 있다. 이처럼, 크론병, 다발성 경화증, 1형 당뇨병, 자가 면역성 간염, 이식 편대 숙주병과 같은 다양한 면역 질환에 대한 중간엽 줄기세포의 효용성 연구가 계속되고 있다. 구체적으로, 건선의 경우 제대혈 유래 중간엽 줄기세포를 이용한 임상 시험이 진행 중이다. 이에, 본 연구진은 세포 채취가 용이하고, 줄기세포가 풍부한 지방 유래 중간엽 줄기세포를 이용한 연구를 진행하는 바이다.


  실험 방법

  환자 별 특성

  환자 A.     

58세 필리핀 남성 (52kg) 1987년 건선 진단을 받았고, 2008년 관절염 진단을 받았다. 그는 얼굴 중앙과 양 발바닥에 홍반과 각질 형성, Nail Pitting (손톱 표면이 움푹 페이는 현상), 모든 손톱, 발톱이 부스러지고, 떨어져나가는 ‘조갑박리증’ 과 함께 팔, 다리, 가슴, 복부 등에 각질이 일어나 이동, 목욕에 큰 어려움을 느꼈다. 지방 줄기세포 임상 실험 참여 전에 사용한 Chobetasol 연고 등 다양한 식물성 추출물을 이용한 차료를 받았지만 뚜렷한 개선은 경험하지 못했다.

 

  환자 B

28세 팔리핀 여성 (45kg) 16살에서 21살 때 까지, 5년간 면역 억제제 (Methotrezate) 매주 복용했다. 25세에 갑상선 기능 항진증 진단을 받았으며, 22살 떼부터 스테로이드 계열 약물의 복용을 멀리해왔지만, 한 달에 한번 꼴로 극심한 통증에 못 이겨 복용하곤 했다. 또한, 면역 억제제 역시 매번 일정한 약효를 보이지는 않는다고 밝혔다.

 

  임상 실험 수행

환자 A 의 경우, 2번의 지방 줄기세포 투여 (실험 참여 당일 (Day 0) 40일 이후 (Day 40))를 진행했고, B 의 경우, 3 (실험 참여 당일 (Day 0) 30일 이후 (Day 30), 71일 이후 (Day 71)). 각각의 투여 간격은 자체 연구 결과에 따라 결정했다.  두 경우 모두 부작용은 나타나지 않았고, 투여 경과를 1년간 관찰하였다.

 

 

  결과

환자 A 는 최초 투여 당일 PASI 지수가 21.6 이었으며, 상당히 많은 각질이 관찰되었고, 오른쪽 무릎이 부풀어올라 통증을 호소했다. 40일 뒤, PASI 지수는 8.9 를 기록했고, 홍반, 피부 딱딱해짐, 각질, 피부 상처 등이 눈에 띄게 감소했다. 이후, PASI 지수는 157일 까지 유지되었으며, 조갑박리증 역시 개선되었다.

한편, 환자 B의 초기 PASI 지수는 9.9 였다. 줄기세포와 복용중인 면역 억제제 등의 약물 간의 효능 간섭을 피하기 위해 최초 추여 23일 전부터 면역 억제제는 복용하지 않았다. 면역 억제제를 복용하지 않은 상태로 줄기세포 투여 직전에 다시 측정한 PASI 지수는 24 이었으며, 조갑박리증이 손톱과 발톱 모두에서 심한 것으로 관찰되었다. 최초 투여 30일 이후, 줄기세포 2차 투여 뒤에 측정한 PASI 지수는 18.3 을 기록했고, 최초 투여 71일 이후 3차 투여 뒤에는 9.4를 기록했으며, 조갑박리증 증상이 현저히 줄어있었다. 231일 뒤, PASI 지수는 8.3을 나타냈다.