The Surprisingly Simple Recipe to Start Growing a Member

How do organisms form limbs in the uterus? Scientists have worked to answer this question not only to deepen our understanding of evolution and embryonic development, but also to help make the dream of regenerating partial or entire limbs a reality.

A team led by geneticists from Harvard Medical School has just taken a step forward on this long road.

As described in their article published on February 5 in Development cellResearchers have identified the special ingredients needed to initiate the creation of limbs in mice and chicks.

“People in the field know about a lot of the proteins that are essential for limb formation, but we found that there were some proteins that we were missing,” said study co-first author ChangHee Lee, a researcher in genetics in the laboratory of Cliff Tabin at HMS.

The team discovered that a combination of just three proteins – Prdm16, Zbtb16 and Lin28a – is necessary and sufficient to transform some non-limb forming stem cells into limb forming cells. A fourth protein, Lin41, speeds up the process.

Part of a family called genetic transcription factors, these proteins activate a handful of genes inside certain cells in embryonic tissue called mesenchyme, the researchers revealed. This change in gene activity is what turns the cells into limb progenitor cells, the team showed.

Limb progenitor cells then bud where a limb will form and provide structure for the future arm, leg, wing or fin.

“We found the proteins that confer ‘membership’ to this subgroup of mesenchymal cells,” Lee said. “Before, people didn’t know how to turn mesenchymal stem cells into limb progenitors. Now we can do that and study early limb differentiation.”

Future work must confirm whether the same transcription factors are at play in human development. Early work is promising, the team said.

It also remains to be discovered what other ingredients need to be added for limb progenitor cells to mature in the connective tissues of the limb, such as tendons, ligaments and the middle layer of skin.

How this work advances stem cell research

This discovery allows scientists for the first time to take mouse fibroblasts – connective tissue cells commonly used to explore how stem cells mature into different tissues – and direct them to limb progenitors.

This work also now allows scientists to keep limb progenitor cells alive in the laboratory for much longer than was previously possible – weeks instead of a day or two. That’s enough time to start really digging into the mechanisms of early limb development, Lee said.

Members of the Tabin lab made all this possible by creating a tool to grow limb progenitor cells in 3D structures, then optimizing nearly 30 cell culture conditions until the cells grew.

The team was excited to finally be able to make limb progenitor cells “survive, proliferate, and, critically, maintain their limb progenitor identity after prolonged culture,” said Tabin, co-senior author, professor of genetics George Jacob and Jacqueline Hazel Leder and leader of the study. Department of Genetics at the Blavatnik Institute at HMS.

The optimal growth parameter set is a more important contribution to the field than 3D scaffolding, Lee said. The team has made the protocols freely available online.

“We tested many conditions to see what the cells like and what they don’t like. We found that they are particularly picky about stiffness,” Lee said. “The only limitation we’ve found so far is that the cells grow so well that they fill the containers we use, which is a good problem.”

Questions that limb development studies could now answer

Developmental and evolutionary biologists and regenerative medicine scientists are now better positioned to answer questions such as:

• The roles that the three genetic transcription factors play in other organ systems and organisms.
• What factors contribute to the later development of limbs, such as fingers and toes?
• What distinguishes the development of the front and rear limbs.
• How this knowledge can inform efforts to regrow different organs to treat injuries or illnesses.

“It is important to understand the fundamental properties of cells that have therapeutic value,” Lee said. “Cultivating and maintaining limb progenitor cells and directing them toward more specific lineages is of fundamental importance for the long-term goal of cell reconstitution in the clinic.”

The work also supports the underdog argument that mammals can serve as useful model organisms for limb regeneration, even though they cannot regrow their limbs after birth.

“Understanding and exploiting mammalian limb progenitors is a first step toward considering mammals as models for amputated limb regeneration, as an alternative to the amphibians and other limb-regenerating creatures studied today,” Tabin said.