Medical Bulletin 06/ March/ 2024

In a collaborative research, scientists from Kyushu University and Harvard Medical School have discovered proteins capable of transforming or "reprogramming" fibroblasts — the predominant cells in skin and connective tissue — into cells possessing characteristics similar to limb progenitor cells.

The findings published in the Journal Developmental Cell enhanced the understanding of limb development and laid the groundwork for regenerative therapy in the future.

Approximately 60 million individuals worldwide are living with limb loss, a consequence of diverse medical issues like tumours, infections, birth defects, or trauma from accidents, as well as natural disasters. Those with limb injuries often resort to synthetic materials and metal prostheses for support. However, numerous researchers are delving into the intricacies of limb development, striving to advance regenerative therapy or natural tissue replacement as a promising treatment option.

“During limb development in the embryo, limb progenitor cells in the limb bud give rise to most of the different limb tissues, such as bone, muscle, cartilage and tendon. It’s therefore important to establish an easy and accessible way of making these cells,” explained Dr. Yuji Atsuta, lead researcher at The Harvard Medical School.

Currently, limb progenitor cells are commonly sourced from embryos, posing ethical dilemmas with human embryos. Alternatively, induced pluripotent stem cells, reprogrammed from adult cells, can be utilized, but they carry risks of cancerous transformation. The new method developed by Atsuta and colleagues, which directly reprograms fibroblast cells into limb progenitor cells and bypasses induced pluripotent stem cells, simplifies the process and reduces costs. It also mitigates the concern of cells turning cancerous, which often occurs with induced pluripotent stem cells.

In the initial phase, researchers examined gene expression in early limb buds of mice and chicken embryos. They found 18 genes, mostly transcription factors, highly expressed in limb buds. Introducing these genes into fibroblasts from mouse embryos led to the cells resembling limb progenitor cells in gene expression. Further experiments identified three essential proteins—Prdm16, Zbtb16, and Lin28a—to reprogram fibroblasts into limb progenitor-like cells, with Lin41 aiding in cell growth and multiplication.

“These reprogrammed cells are not only molecular mimics; we have confirmed their potential to develop into specialized limb tissues, both in laboratory dishes (in vitro) and also in living organisms (in vivo),” said Atsuta. “Testing in vivo was particularly challenging, as we had to transplant the reprogrammed mouse cells into the limb buds of chicken embryos.”

Reference: Yuji Atsuta 8, ChangHee Lee 8, Alan R. Rodrigues 8, Charlotte Colle, Reiko R. Tomizawa, Ernesto G. Lujan,Patrick Tschopp, Laura Galan, Meng Zhu, Joshua M. Gorham, Jean-Pierre Vannier, Christine E. Seidman, Jonathan G. Seidman, Marian A. Ros, Olivier Pourquié, Clifford J. Tabin 9; JOURNAL: Developmental Cell; DOI: 10.1016/j.devcel.2023.12.010

Cell crowding in early human embryos influences cell identity decisions, finds a new culture system

New research conducted by the Institute and the Wellcome-MRC Cambridge Stem Cell Institute has developed a cell culture system that differentiates human pluripotent stem cells to amniotic ectoderm and surface ectoderm based on cell density.

Published in Journal Science Advances, the research revealed that cell density in early human embryos affects whether cells become extra-embryonic or contribute to the embryo, forming skin, hair, and nails.

The amniotic ectoderm, forming the embryo's surrounding membrane, provides crucial signals for human embryo development, yet its origin remains partially understood. Surface ectoderm, responsible for skin and related structures, shares similarities with amniotic ectoderm but is not fully elucidated in its development. Despite differences, both tissues exhibit early specialization and common biological features.

Dr. Shota Nakanoh devised tailored culture conditions for human pluripotent stem cells (hPSCs), discovering that specific supplements prompted their differentiation into amniotic ectoderm. Utilizing a co-culture method, he verified the ability of these cells to initiate gastrulation. Single-cell RNA sequencing analysis indicated a differentiation pathway from surface ectoderm to amniotic ectoderm.

Comparison with primate embryo data confirmed cultured cells' similarity to embryonic tissues. Cell density determined cell fate, favoring amniotic ectoderm under specific conditions and surface ectoderm under high density. The study also found cells resembling extra-embryonic mesoderm. Accurate amnion formation is vital for mimicking human embryo development in stem cell-based models, allowing study beyond technical and regulatory limitations on human embryo research.

“We have only recently begun to explore the generation of amnion during human development. These findings advance our understanding on how to generate extra-embryonic cells in the lab in vitro, and sheds light on the mechanisms that drive the formation of cell types at the stages that correspond to the ‘black box’ of human development. Given the growing interest in using stem cell embryo models as proxies of human embryos, this work provides more knowledge for the generation of successful integrated models.” said Dr Teresa Rayon, group leader in the Institute’s Epigenetics research programme.

“Our culture system also generates extra-embryonic mesoderm, another tissue not studied well in human embryos. It will provide better understanding of human development and could improve our knowledge about diseases affecting first step of foetal life. This work also opens the door for new studies regarding the role of cellular density in cell fate decision.” concluded Prof. Vallier, Professor of Stem Cells in Regenerative Therapies at the Berlin Institute of Health at Charité (BIH).

Reference: SHOTA NAKANOH, KENDIG SHAM, SABITRI GHIMIRE, IRINA MOHORIANU, TERESA RAYON, LUDOVIC VALLIER; JOURNAL: Science Advances; DOI: 10.1126/sciadv.adh7748

Exploring the genome of male breast cancer

According to a study conducted by researchers at Weill Cornell Medicine, distinct genetic alterations in the tumour genome of male breast cancer could indicate potential treatment targets. The study marked the first whole-genome sequencing analysis of male breast cancer, examining the comprehensive DNA landscape.

Published in the Journal Modern Pathology, the study uncovered gene mutations and molecular profiles involved in male breast cancer that could impact diagnosis and treatment.

Male breast cancer, which represents less than 1 percent of all breast cancer cases each year, has increased at a much faster rate than in women over the last 40 years. Unaware of the risk, breast cancer in males tends to be diagnosed at more advanced stages and hence, have poorer treatment outcomes.

For the study, researchers detected mutations in multiple genes within tumor samples from 10 patients, known to drive cancer growth. They also identified structural variants—places in the genome where DNA has broken and rearranged—that impact five other cancer-associated genes. Two men had variations in the BRCA2 gene that impairs DNA repair, a common cause of breast cancer in women. Along with an extended study group of 18 additional tumor samples, about 21 percent of the tumors had 10 to 20 excess copies of the FGFR1 gene, which is linked to treatment-resistant tumors in some women with breast cancer.

Cancer therapies are readily available to target the genetic variations identified in 8 of the 10 men, opening new pathways for treatment. For example, drugs such as immunotherapy and PARP inhibitors might be effective for men with BRCA2 gene variations and a high number of tumor mutations. Cancer-triggering rearrangements in the NTRK1 gene may respond to drugs called kinase inhibitors. In addition, the gene mutations identified may lead to the discovery of new targeted therapies.

“Though larger studies will need to done to confirm the research results, the novel findings suggest that tailoring treatments for this under-studied male patient population will be necessary,” said the authors.

Reference: Dr. Majd Al Assaad, Dr. Juan Miguel Mosquera, Sandra and Edward Meyer; JOURNAL: Modern Pathology